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Hoffmann S, Seeger T. Advances in human induced pluripotent stem cell (hiPSC)-based disease modelling in cardiogenetics. MED GENET-BERLIN 2025; 37:137-146. [PMID: 40207041 PMCID: PMC11976404 DOI: 10.1515/medgen-2025-2009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/11/2025]
Abstract
Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.
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Affiliation(s)
- Sandra Hoffmann
- University Hospital HeidelbergInstitute of Human GeneticsHeidelbergGermany
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2
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Kim J, Nam Y, Jeon D, Choi Y, Choi S, Hong CP, Kim S, Jung H, Park N, Sohn Y, Rim YA, Ju JH. Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout. Exp Mol Med 2025; 57:686-699. [PMID: 40087529 PMCID: PMC11958689 DOI: 10.1038/s12276-025-01422-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 10/20/2024] [Accepted: 12/16/2024] [Indexed: 03/17/2025] Open
Abstract
Hypoimmunogenic universal induced pluripotent stemn (iPS) cells were generated through the targeted disruption of key genes, including human leukocyte antigen (HLA)-A, HLA-B and HLA-DR alpha (DRA), using the CRISPR-Cas9 system. This approach aimed to minimize immune recognition and enhance the potential of iPS cells for allogeneic therapy. Heterozygous iPS cells were used for guide RNA design and validation to facilitate the knockout (KO) of the HLA-A, HLA-B and HLA-DRA genes. The electroporation of iPS cells using the selected guide RNAs enabled the generation of triple-KO iPS cells, followed by single-cell cloning for clone selection. Clone A7, an iPS cell with targeted KOs of the HLA-A, HLA-B and HLA-DRA genes, was identified as the final candidate. Messenger RNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4, sex-determining region Y box 2, Krüppel-like factor 4, Lin-28 homolog A and Nanog homeobox, while protein expression assays confirmed the presence of octamer-binding transcription factor 4, stage-specific embryonic antigen 4, Nanog homeobox and tumor rejection antigen 1-60. A karyotype examination revealed no anomalies, and three-germ layer differentiation assays confirmed the differentiation potential. After interferon gamma stimulation, the gene-corrected clone A7 lacked HLA-A, HLA-B and HLA-DR protein expression. Immunogenicity testing further confirmed the hypoimmunogenicity of clone A7, which was evidenced by the absence of proliferation in central memory T cells and effector memory T cells. In conclusion, clone A7, a triple-KO iPS cell clone that demonstrates immune evasion properties, retained its intrinsic iPS cell characteristics and exhibited no immunogenicity.
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Affiliation(s)
| | - Yoojun Nam
- YiPSCELL Inc., Seoul, Republic of Korea
- Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, Republic of Korea
| | | | | | | | | | | | | | | | - Yeowon Sohn
- Department of Biohealth Regulatory Science, Sungkyunkwan University, Suwon, Republic of Korea
| | - Yeri Alice Rim
- CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
| | - Ji Hyeon Ju
- YiPSCELL Inc., Seoul, Republic of Korea.
- CiSTEM Laboratory, Convergent Research Consortium for Immunologic Disease, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary's Hospital, Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
- Department of Biomedicine and Health Sciences, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
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Ye J, Boileau RM, Parchem RJ, Judson-Torres RL, Blelloch R. The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells. Stem Cells 2025; 43:sxae080. [PMID: 40037390 PMCID: PMC11879289 DOI: 10.1093/stmcls/sxae080] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Accepted: 10/24/2024] [Indexed: 03/06/2025]
Abstract
The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell (ESC)-specific cell cycle regulating family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their ESC microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state.
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Affiliation(s)
- Julia Ye
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
| | - Ryan M Boileau
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
| | - Ronald J Parchem
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, United States
| | - Robert L Judson-Torres
- Huntsman Cancer Institute, The University of Utah, Salt Lake City, UT 84112, United States
- Department of Dermatology, The University of Utah, Salt Lake City, UT 84112, United States
| | - Robert Blelloch
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California at San Francisco, San Francisco, CA 94143, United States
- Center for Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143, United States
- Department of Urology, University of California at San Francisco, San Francisco, CA 94143, United States
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Huang Y, Zhang Y, Wang Z, Miao L, Tan P, Guan Y, Ran Y, Feng X, Wang Y, Guo Y, Guo X. Modified mRNA-based gene editing reveals sarcomere-based regulation of gene expression in human induced-pluripotent stem cell-derived cardiomyocytes. Int Immunopharmacol 2024; 143:113378. [PMID: 39423657 DOI: 10.1016/j.intimp.2024.113378] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2024] [Revised: 09/17/2024] [Accepted: 10/07/2024] [Indexed: 10/21/2024]
Abstract
Mutations in genes coding sarcomere components are the major causes of human inherited cardiomyopathy. Genome editing is widely applied to genetic modification of human pluripotent stem cells (hPSCs) before hPSCs were differentiated into cardiomyocytes to model cardiomyopathy. Whether genetic mutations influence the early hPSC differentiation process or solely the terminally differentiated cardiomyocytes during cardiac pathogenesis remains challenging to distinguish. To solve this problem, here we harnessed chemically modified mRNA (modRNA) and synthetic single-guide RNA to develop an efficient genome editing approach in hPSC-derived cardiomyocytes (hPSC-CMs). We showed that modRNA-based CRISPR/Cas9 mutagenesis of TNNT2, the coding gene for cardiac troponin T, results in sarcomere disassembly and contractile dysfunction in hPSC-CMs. These structural and functional phenotypes were associated with profound downregulation of oxidative phosphorylation genes and upregulation of cardiac stress markers NPPA and NPPB. These data confirmed that sarcomeres regulate gene expression in hPSC-CMs and highlighted the RNA technology as a powerful tool to achieve stage-specific genome editing during hPSC differentiation.
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Affiliation(s)
- Yuqing Huang
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yueyang Zhang
- School of Basic Medical Sciences, Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China
| | - Ze Wang
- School of Basic Medical Sciences, Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China
| | - Lei Miao
- State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China; Beijing Key Laboratory of Molecular Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China
| | - Pingping Tan
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yuting Guan
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yuqing Ran
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xing Feng
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yijia Wang
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yuxuan Guo
- School of Basic Medical Sciences, Institute of Cardiovascular Sciences, Peking University Health Science Center, Beijing, China; State Key Laboratory of Vascular Homeostasis and Remodeling, Peking University, Beijing, China; Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing, China.
| | - Xiaoling Guo
- Basic Medical Research Center, the Second Affiliated Hospital and Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
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Mir A, Zhu A, Lau R, Barr N, Sheikh Z, Acuna D, Dayal A, Hibino N. Applications, Limitations, and Considerations of Clinical Trials in a Dish. Bioengineering (Basel) 2024; 11:1096. [PMID: 39593756 PMCID: PMC11591410 DOI: 10.3390/bioengineering11111096] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2024] [Revised: 10/26/2024] [Accepted: 10/28/2024] [Indexed: 11/28/2024] Open
Abstract
Recent advancements in biotechnology forged the path for clinical trials in dish (CTiDs) to advance as a popular method of experimentation in biomedicine. CTiDs play a fundamental role in translational research through technologies such as induced pluripotent stem cells, whole genome sequencing, and organs-on-a-chip. In this review, we explore advancements that enable these CTiD biotechnologies and their applications in animal testing, disease modeling, and space radiation technologies. Furthermore, this review dissects the advantages and disadvantages of CTiDs, as well as their regulatory considerations. Lastly, we evaluate the challenges that CTiDs pose and the role of CTiDs in future experimentation.
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Affiliation(s)
- Amatullah Mir
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Angie Zhu
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Rico Lau
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Nicolás Barr
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Zyva Sheikh
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Diana Acuna
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Anuhya Dayal
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
| | - Narutoshi Hibino
- Section of Cardiac Surgery, Department of Surgery, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637, USA; (A.M.); (A.Z.); (R.L.); (N.B.); (Z.S.); (D.A.); (A.D.)
- Pediatric Cardiac Surgery, Advocate Children’s Hospital, 4440 W 95th St., Oak Lawn, IL 60453, USA
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Villafranco J, Martínez-Ramírez G, Magaña-Maldonado R, González-Ruvalcaba AP, López-Ornelas A, Velasco I, Becerril-Villanueva E, Pavón L, Estudillo E, Pérez-Sánchez G. The use of induced pluripotent stem cells as a platform for the study of depression. Front Psychiatry 2024; 15:1470642. [PMID: 39444629 PMCID: PMC11496182 DOI: 10.3389/fpsyt.2024.1470642] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Accepted: 09/17/2024] [Indexed: 10/25/2024] Open
Abstract
The neurobiological mechanisms underlying major depressive disorder (MDD) remain largely unexplored due to the limited availability of study models in humans. Induced pluripotent stem cells (iPSCs) have overcome multiple limitations of retrospective clinical studies, contributing to a more detailed understanding of the molecular pathways that presumably contribute to the manifestation of depression. Despite the significant progress made by these study models, there are still more formidable challenges that will eventually be addressed by these platforms, as further studies may eventually emerge. This review will examine the most recent advances in the comprehension of depression by using human neurons and non-neuronal cells derived from induced pluripotent stem cells of patients with depression. This study highlights the importance of using these platforms to increase our knowledge of depression and address this psychiatric disorder more efficiently.
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Affiliation(s)
- Javier Villafranco
- Laboratorio de Reprogramación Celular, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Ciudad de México, Mexico
- Laboratorio de Psicoinmunología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, Ciudad de México, Mexico
- Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (UNAM), Tlalnepantla, Mexico
| | - Gabriela Martínez-Ramírez
- Laboratorio de Psicoinmunología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, Ciudad de México, Mexico
- Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México (UNAM), Tlalnepantla, Mexico
| | - Roxana Magaña-Maldonado
- Laboratorio de Reprogramación Celular, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Ciudad de México, Mexico
| | - Anna Paola González-Ruvalcaba
- Laboratorio de Reprogramación Celular, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Ciudad de México, Mexico
| | - Adolfo López-Ornelas
- División de Investigación, Hospital Juárez de México, Mexico City, Mexico
- Hospital Nacional Homeopático, Hospitales Federales de Referencia, Mexico City, Mexico
| | - Iván Velasco
- Laboratorio de Reprogramación Celular, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Ciudad de México, Mexico
- Instituto de Fisiología Celular - Neurociencias, Universidad Nacional Autónoma de México, México City, Mexico
| | - Enrique Becerril-Villanueva
- Laboratorio de Psicoinmunología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, Ciudad de México, Mexico
| | - Lenin Pavón
- Laboratorio de Psicoinmunología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, Ciudad de México, Mexico
| | - Enrique Estudillo
- Laboratorio de Reprogramación Celular, Instituto Nacional de Neurología y Neurocirugía Manuel Velasco Suárez, Ciudad de México, Mexico
| | - Gilberto Pérez-Sánchez
- Laboratorio de Psicoinmunología, Instituto Nacional de Psiquiatría Ramón de la Fuente Muñíz, Ciudad de México, Mexico
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Ye J, Boileau RM, Parchem RJ, Judson-Torres RL, Blelloch R. The miR-290 and miR-302 clusters are essential for reprogramming of fibroblasts to induced pluripotent stem cells. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.09.02.610895. [PMID: 39282363 PMCID: PMC11398367 DOI: 10.1101/2024.09.02.610895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 10/25/2024]
Abstract
The miR-290 and miR-302 clusters of microRNAs are highly expressed in naïve and primed pluripotent stem cells, respectively. Ectopic expression of the embryonic stem cell-specific cell cycle regulating (ESCC) family of microRNAs arising from these two clusters dramatically enhances the reprogramming of both mouse and human somatic cells to induced pluripotency. Here, we used genetic knockouts to dissect the requirement for the miR-290 and miR-302 clusters during the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs) with retrovirally introduced Oct4, Sox2, and Klf4. Knockout of either cluster alone did not negatively impact the efficiency of reprogramming. Resulting cells appeared identical to their embryonic stem cell microRNA cluster knockout counterparts. In contrast, the combined loss of both clusters blocked the formation of iPSCs. While rare double knockout clones could be isolated, they showed a dramatically reduced proliferation rate, a persistent inability to fully silence the exogenously introduced pluripotency factors, and a transcriptome distinct from individual miR-290 or miR-302 mutant ESC and iPSCs. Taken together, our data show that miR-290 and miR-302 are essential yet interchangeable in reprogramming to the induced pluripotent state. Impact Statement The process by which somatic cell reprogramming yields induced pluripotent stem cells (iPSCs) is incompletely understood. MicroRNAs from the miR-290 and miR-302 clusters have been shown to greatly increase reprogramming efficiency, but their requirement in the process has not been studied. Here, we examine this requirement by genetically removing the miRNA clusters in somatic cells. We discover that somatic cells lacking either, but not both, of these miRNA clusters can form iPSC cells. This work thus provides new important insight into mechanisms underlying reprogramming to pluripotency.
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Affiliation(s)
- Julia Ye
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
| | - Ryan M. Boileau
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
| | - Ronald J. Parchem
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Robert L. Judson-Torres
- Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA
- Department of Dermatology, University of Utah, Salt Lake City, UT 84112, USA
| | - Robert Blelloch
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California, 94143, USA
- Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California, 94143, USA
- Department of Urology, University of California, San Francisco, San Francisco, California, 94143, USA
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Risato G, Brañas Casas R, Cason M, Bueno Marinas M, Pinci S, De Gaspari M, Visentin S, Rizzo S, Thiene G, Basso C, Pilichou K, Tiso N, Celeghin R. In Vivo Approaches to Understand Arrhythmogenic Cardiomyopathy: Perspectives on Animal Models. Cells 2024; 13:1264. [PMID: 39120296 PMCID: PMC11311808 DOI: 10.3390/cells13151264] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Revised: 07/23/2024] [Accepted: 07/24/2024] [Indexed: 08/10/2024] Open
Abstract
Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac disorder characterized by the gradual replacement of cardiomyocytes with fibrous and adipose tissue, leading to ventricular wall thinning, chamber dilation, arrhythmias, and sudden cardiac death. Despite advances in treatment, disease management remains challenging. Animal models, particularly mice and zebrafish, have become invaluable tools for understanding AC's pathophysiology and testing potential therapies. Mice models, although useful for scientific research, cannot fully replicate the complexity of the human AC. However, they have provided valuable insights into gene involvement, signalling pathways, and disease progression. Zebrafish offer a promising alternative to mammalian models, despite the phylogenetic distance, due to their economic and genetic advantages. By combining animal models with in vitro studies, researchers can comprehensively understand AC, paving the way for more effective treatments and interventions for patients and improving their quality of life and prognosis.
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Affiliation(s)
- Giovanni Risato
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
- Department of Biology, University of Padua, I-35131 Padua, Italy;
- Department of Women’s and Children’s Health, University of Padua, I-35128 Padua, Italy;
| | | | - Marco Cason
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Maria Bueno Marinas
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Serena Pinci
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Monica De Gaspari
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Silvia Visentin
- Department of Women’s and Children’s Health, University of Padua, I-35128 Padua, Italy;
| | - Stefania Rizzo
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Gaetano Thiene
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Cristina Basso
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Kalliopi Pilichou
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
| | - Natascia Tiso
- Department of Biology, University of Padua, I-35131 Padua, Italy;
| | - Rudy Celeghin
- Department of Cardio-Thoraco-Vascular Sciences and Public Health, University of Padua, I-35128 Padua, Italy; (G.R.); (M.C.); (M.B.M.); (S.P.); (M.D.G.); (S.R.); (G.T.); (C.B.); (K.P.); (R.C.)
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Portela-Lomba M, Simón D, Callejo-Móstoles M, de la Fuente G, Fernández de Sevilla D, García-Escudero V, Moreno-Flores MT, Sierra J. Generation of functional neurons from adult human mucosal olfactory ensheathing glia by direct lineage conversion. Cell Death Dis 2024; 15:478. [PMID: 38961086 PMCID: PMC11222439 DOI: 10.1038/s41419-024-06862-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2023] [Revised: 06/21/2024] [Accepted: 06/25/2024] [Indexed: 07/05/2024]
Abstract
A recent approach to promote central nervous system (CNS) regeneration after injury or disease is direct conversion of somatic cells to neurons. This is achieved by transduction of viral vectors that express neurogenic transcription factors. In this work we propose adult human mucosal olfactory ensheathing glia (hmOEG) as a candidate for direct reprogramming to neurons due to its accessibility and to its well-characterized neuroregenerative capacity. After induction of hmOEG with the single neurogenic transcription factor NEUROD1, the cells under study exhibited morphological and immunolabeling neuronal features, fired action potentials and expressed glutamatergic and GABAergic markers. In addition, after engraftment of transduced hmOEG cells in the mouse hippocampus, these cells showed specific neuronal labeling. Thereby, if we add to the neuroregenerative capacity of hmOEG cultures the conversion to neurons of a fraction of their population through reprogramming techniques, the engraftment of hmOEG and hmOEG-induced neurons could be a procedure to enhance neural repair after central nervous system injury.
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Affiliation(s)
- María Portela-Lomba
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain
- Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA
| | - Diana Simón
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain
| | - Marta Callejo-Móstoles
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - Gemma de la Fuente
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - David Fernández de Sevilla
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - Vega García-Escudero
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - M Teresa Moreno-Flores
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain.
| | - Javier Sierra
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain.
- School of Medicine, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Spain.
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10
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Tsuchida M, Hasegawa M, Miki K, Miyagawa S, Kashino K. Mechanical Performance of Engineered Heart Tissue Can Be Measured with POC-Based Video Analysis. ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY. ANNUAL INTERNATIONAL CONFERENCE 2024; 2024:1-5. [PMID: 40039845 DOI: 10.1109/embc53108.2024.10781552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/06/2025]
Abstract
Recently, research aiming at elucidation of causes and therapies of diseases using artificial tissues derived from disease-specific induced pluripotent stem cells (iPSCs) has been active. An important part of this approach is to quantitatively measure the functional health or performance of the artificial tissue of interest. Specifically, in the engineered heart tissue (EHT), it is very important to measure its contraction and relaxation forces. To this end, we develop a method based on the Phase-only Correlation (POC) for measuring contractile and diastolic forces by culturing cardiomyocytes on a pair of pillars with known mechanical properties and capturing video of tissue movement in response to a cyclic stimulus using a fluorescence microscope. The method tracks the tissue movement over the frames and then determines its maximum deflection and frequency by fitting the detected deflection to a sine function. We show that our method enables the measurement of the mechanical performance of the EHT. Accuracy analysis reveals that our method exhibits better accuracy compared to the known HOG-based method.
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11
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Sabetkish S, Currie P, Meagher L. Recent trends in 3D bioprinting technology for skeletal muscle regeneration. Acta Biomater 2024; 181:46-66. [PMID: 38697381 DOI: 10.1016/j.actbio.2024.04.038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2023] [Revised: 04/24/2024] [Accepted: 04/25/2024] [Indexed: 05/05/2024]
Abstract
Skeletal muscle is a pro-regenerative tissue, that utilizes a tissue-resident stem cell system to effect repair upon injury. Despite the demonstrated efficiency of this system in restoring muscle mass after many acute injuries, in conditions of severe trauma such as those evident in volumetric muscle loss (VML) (>20 % by mass), this self-repair capability is unable to restore tissue architecture, requiring interventions which currently are largely surgical. As a possible alternative, the generation of artificial muscle using tissue engineering approaches may also be of importance in the treatment of VML and muscle diseases such as dystrophies. Three-dimensional (3D) bioprinting has been identified as a promising technique for regeneration of the complex architecture of skeletal muscle. This review discusses existing treatment strategies following muscle damage, recent progress in bioprinting techniques, the bioinks used for muscle regeneration, the immunogenicity of scaffold materials, and in vitro and in vivo maturation techniques for 3D bio-printed muscle constructs. The pros and cons of these bioink formulations are also highlighted. Finally, we present the current limitations and challenges in the field and critical factors to consider for bioprinting approaches to become more translationa and to produce clinically relevant engineered muscle. STATEMENT OF SIGNIFICANCE: This review discusses the physiopathology of muscle injuries and existing clinical treatment strategies for muscle damage, the types of bioprinting techniques that have been applied to bioprinting of muscle, and the bioinks commonly used for muscle regeneration. The pros and cons of these bioinks are highlighted. We present a discussion of existing gaps in the literature and critical factors to consider for the translation of bioprinting approaches and to produce clinically relevant engineered muscle. Finally, we provide insights into what we believe will be the next steps required before the realization of the application of tissue-engineered muscle in humans. We believe this manuscript is an insightful, timely, and instructive review that will guide future muscle bioprinting research from a fundamental construct creation approach, down a translational pathway to achieve the desired impact in the clinic.
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Affiliation(s)
- Shabnam Sabetkish
- Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC 3800, Australia
| | - Peter Currie
- Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC 3800, Australia
| | - Laurence Meagher
- Department of Materials Science and Engineering, Monash University, Clayton, Victoria, Australia; ARC Training Centre for Cell and Tissue Engineering Technologies, Monash University, Clayton, VIC 3800, Australia.
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12
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Chen H, Yan G, Wen MH, Brooks KN, Zhang Y, Huang PS, Chen TY. Advancements and Practical Considerations for Biophysical Research: Navigating the Challenges and Future of Super-resolution Microscopy. CHEMICAL & BIOMEDICAL IMAGING 2024; 2:331-344. [PMID: 38817319 PMCID: PMC11134610 DOI: 10.1021/cbmi.4c00019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/10/2024] [Revised: 04/06/2024] [Accepted: 04/10/2024] [Indexed: 06/01/2024]
Abstract
The introduction of super-resolution microscopy (SRM) has significantly advanced our understanding of cellular and molecular dynamics, offering a detailed view previously beyond our reach. Implementing SRM in biophysical research, however, presents numerous challenges. This review addresses the crucial aspects of utilizing SRM effectively, from selecting appropriate fluorophores and preparing samples to analyzing complex data sets. We explore recent technological advancements and methodological improvements that enhance the capabilities of SRM. Emphasizing the integration of SRM with other analytical methods, we aim to overcome inherent limitations and expand the scope of biological insights achievable. By providing a comprehensive guide for choosing the most suitable SRM methods based on specific research objectives, we aim to empower researchers to explore complex biological processes with enhanced precision and clarity, thereby advancing the frontiers of biophysical research.
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Affiliation(s)
- Huanhuan Chen
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Guangjie Yan
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Meng-Hsuan Wen
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Kameron N. Brooks
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Yuteng Zhang
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Pei-San Huang
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
| | - Tai-Yen Chen
- Department of Chemistry, University of Houston, Houston, Texas 77204, United States
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13
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Vo QD, Saito Y, Ida T, Nakamura K, Yuasa S. The use of artificial intelligence in induced pluripotent stem cell-based technology over 10-year period: A systematic scoping review. PLoS One 2024; 19:e0302537. [PMID: 38771829 PMCID: PMC11108174 DOI: 10.1371/journal.pone.0302537] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2024] [Accepted: 04/09/2024] [Indexed: 05/23/2024] Open
Abstract
BACKGROUND Stem cell research, particularly in the domain of induced pluripotent stem cell (iPSC) technology, has shown significant progress. The integration of artificial intelligence (AI), especially machine learning (ML) and deep learning (DL), has played a pivotal role in refining iPSC classification, monitoring cell functionality, and conducting genetic analysis. These enhancements are broadening the applications of iPSC technology in disease modelling, drug screening, and regenerative medicine. This review aims to explore the role of AI in the advancement of iPSC research. METHODS In December 2023, data were collected from three electronic databases (PubMed, Web of Science, and Science Direct) to investigate the application of AI technology in iPSC processing. RESULTS This systematic scoping review encompassed 79 studies that met the inclusion criteria. The number of research studies in this area has increased over time, with the United States emerging as a leading contributor in this field. AI technologies have been diversely applied in iPSC technology, encompassing the classification of cell types, assessment of disease-specific phenotypes in iPSC-derived cells, and the facilitation of drug screening using iPSC. The precision of AI methodologies has improved significantly in recent years, creating a foundation for future advancements in iPSC-based technologies. CONCLUSIONS Our review offers insights into the role of AI in regenerative and personalized medicine, highlighting both challenges and opportunities. Although still in its early stages, AI technologies show significant promise in advancing our understanding of disease progression and development, paving the way for future clinical applications.
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Affiliation(s)
- Quan Duy Vo
- Faculty of Medicine, Department of Cardiovascular Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
- Faculty of Medicine, Nguyen Tat Thanh University, Ho Chi Minh City, Viet Nam
| | - Yukihiro Saito
- Department of Cardiovascular Medicine, Okayama University Hospital, Okayama, Japan
| | - Toshihiro Ida
- Faculty of Medicine, Department of Cardiovascular Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
| | - Kazufumi Nakamura
- Faculty of Medicine, Department of Cardiovascular Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
| | - Shinsuke Yuasa
- Faculty of Medicine, Department of Cardiovascular Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan
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14
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Yin X, Li Q, Shu Y, Wang H, Thomas B, Maxwell JT, Zhang Y. Exploiting urine-derived induced pluripotent stem cells for advancing precision medicine in cell therapy, disease modeling, and drug testing. J Biomed Sci 2024; 31:47. [PMID: 38724973 PMCID: PMC11084032 DOI: 10.1186/s12929-024-01035-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 04/30/2024] [Indexed: 05/12/2024] Open
Abstract
The field of regenerative medicine has witnessed remarkable advancements with the emergence of induced pluripotent stem cells (iPSCs) derived from a variety of sources. Among these, urine-derived induced pluripotent stem cells (u-iPSCs) have garnered substantial attention due to their non-invasive and patient-friendly acquisition method. This review manuscript delves into the potential and application of u-iPSCs in advancing precision medicine, particularly in the realms of drug testing, disease modeling, and cell therapy. U-iPSCs are generated through the reprogramming of somatic cells found in urine samples, offering a unique and renewable source of patient-specific pluripotent cells. Their utility in drug testing has revolutionized the pharmaceutical industry by providing personalized platforms for drug screening, toxicity assessment, and efficacy evaluation. The availability of u-iPSCs with diverse genetic backgrounds facilitates the development of tailored therapeutic approaches, minimizing adverse effects and optimizing treatment outcomes. Furthermore, u-iPSCs have demonstrated remarkable efficacy in disease modeling, allowing researchers to recapitulate patient-specific pathologies in vitro. This not only enhances our understanding of disease mechanisms but also serves as a valuable tool for drug discovery and development. In addition, u-iPSC-based disease models offer a platform for studying rare and genetically complex diseases, often underserved by traditional research methods. The versatility of u-iPSCs extends to cell therapy applications, where they hold immense promise for regenerative medicine. Their potential to differentiate into various cell types, including neurons, cardiomyocytes, and hepatocytes, enables the development of patient-specific cell replacement therapies. This personalized approach can revolutionize the treatment of degenerative diseases, organ failure, and tissue damage by minimizing immune rejection and optimizing therapeutic outcomes. However, several challenges and considerations, such as standardization of reprogramming protocols, genomic stability, and scalability, must be addressed to fully exploit u-iPSCs' potential in precision medicine. In conclusion, this review underscores the transformative impact of u-iPSCs on advancing precision medicine and highlights the future prospects and challenges in harnessing this innovative technology for improved healthcare outcomes.
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Affiliation(s)
- Xiya Yin
- Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, China
| | - Qingfeng Li
- Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
| | - Yan Shu
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Hongbing Wang
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Baltimore, MD, USA
| | - Biju Thomas
- Keck School of Medicine, Roski Eye Institute, University of Southern California, Los Angeles, CA, 90033, USA
| | - Joshua T Maxwell
- Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA
| | - Yuanyuan Zhang
- Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC, USA.
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15
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Kantawala B, Shariff S, Ramadan N, Fawaz V, Hassan Y, Mugisha N, Yenkoyan K, Nazir A, Uwishema O. Revolutionizing neurotherapeutics: blood-brain barrier-on-a-chip technologies for precise drug delivery. Ann Med Surg (Lond) 2024; 86:2794-2804. [PMID: 38694300 PMCID: PMC11060226 DOI: 10.1097/ms9.0000000000001887] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2023] [Accepted: 02/23/2024] [Indexed: 05/04/2024] Open
Abstract
Introduction The blood-brain barrier (BBB) is a critical neurovascular unit regulating substances' passage from the bloodstream to the brain. Its selective permeability poses significant challenges in drug delivery for neurological disorders. Conventional methods often fail due to the BBB's complex structure. Aim The study aims to shed light on their pivotal role in revolutionizing neurotherapeutics and explores the transformative potential of BBB-on-a-Chip technologies in drug delivery research to comprehensively review BBB-on-a-chip technologies, focusing on their design, and substantiate advantages over traditional models. Methods A detailed analysis of existing literature and experimental data pertaining to BBB-on-a-Chip technologies was conducted. Various models, their physiological relevance, and innovative design considerations were examined through databases like Scopus, EbscoHost, PubMed Central, and Medline. Case studies demonstrating enhanced drug transport through BBB-on-a-Chip models were also reviewed, highlighting their potential impact on neurological disorders. Results BBB-on-a-Chip models offer a revolutionary approach, accurately replicating BBB properties. These microphysiological systems enable high-throughput screening, real-time monitoring of drug transport, and precise localization of drugs. Case studies demonstrate their efficacy in enhancing drug penetration, offering potential therapies for diseases like Parkinson's and Alzheimer's. Conclusion BBB-on-a-Chip models represent a transformative milestone in drug delivery research. Their ability to replicate BBB complexities, offer real-time monitoring, and enhance drug transport holds immense promise for neurological disorders. Continuous research and development are imperative to unlock BBB-on-a-Chip models' full potential, ushering in a new era of targeted, efficient, and safer drug therapies for challenging neurological conditions.
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Affiliation(s)
- Burhan Kantawala
- Oli Health Magazine Organization, Research and Education
- Neuroscience Laboratory, Cobrain Centre
| | - Sanobar Shariff
- Oli Health Magazine Organization, Research and Education
- Neuroscience Laboratory, Cobrain Centre
| | - Nagham Ramadan
- Oli Health Magazine Organization, Research and Education
- Faculty of Medicine
| | - Violette Fawaz
- Oli Health Magazine Organization, Research and Education
- Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon
| | - Youmna Hassan
- Oli Health Magazine Organization, Research and Education
- Faculty of Medicine and Surgery, Ahfad University for Women, Omdurman, Sudan
| | - Nadine Mugisha
- Oli Health Magazine Organization, Research and Education
- Faculty of Global Surgery, University of Global Health Equity, Kigali, Rwanda
| | - Konstantin Yenkoyan
- Neuroscience Laboratory, Cobrain Centre
- Department of Biochemistry, Yerevan State Medical University named after Mkhitar Heratsi, Yerevan, Armenia
| | - Abubakar Nazir
- Oli Health Magazine Organization, Research and Education
- Department of Medicine, King Edward Medical University, Lahore, Pakistan
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16
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Diaw SH, Delcambre S, Much C, Ott F, Kostic VS, Gajos A, Münchau A, Zittel S, Busch H, Grünewald A, Klein C, Lohmann K. DYT-THAP1: exploring gene expression in fibroblasts for potential biomarker discovery. Neurogenetics 2024; 25:141-147. [PMID: 38498291 DOI: 10.1007/s10048-024-00752-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Accepted: 03/04/2024] [Indexed: 03/20/2024]
Abstract
Dystonia due to pathogenic variants in the THAP1 gene (DYT-THAP1) shows variable expressivity and reduced penetrance of ~ 50%. Since THAP1 encodes a transcription factor, modifiers influencing this variability likely operate at the gene expression level. This study aimed to assess the transferability of differentially expressed genes (DEGs) in neuronal cells related to pathogenic variants in the THAP1 gene, which were previously identified by transcriptome analyses. For this, we performed quantitative (qPCR) and Digital PCR (dPCR) in cultured fibroblasts. RNA was extracted from THAP1 manifesting (MMCs) and non-manifesting mutation carriers (NMCs) as well as from healthy controls. The expression profiles of ten of 14 known neuronal DEGs demonstrated differences in fibroblasts between these three groups. This included transcription factors and targets (ATF4, CLN3, EIF2A, RRM1, YY1), genes involved in G protein-coupled receptor signaling (BDKRB2, LPAR1), and a gene linked to apoptosis and DNA replication/repair (CRADD), which all showed higher expression levels in MMCs and NMCs than in controls. Moreover, the analysis of genes linked to neurological disorders (STXBP1, TOR1A) unveiled differences in expression patterns between MMCs and controls. Notably, the genes CUEDC2, DRD4, ECH1, and SIX2 were not statistically significantly differentially expressed in fibroblast cultures. With > 70% of the tested genes being DEGs also in fibroblasts, fibroblasts seem to be a suitable model for DYT-THAP1 research despite some restrictions. Furthermore, at least some of these DEGs may potentially also serve as biomarkers of DYT-THAP1 and influence its penetrance and expressivity.
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Affiliation(s)
| | - Sylvie Delcambre
- Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, L-4362, Luxembourg
| | - Christoph Much
- Institute of Neurogenetics, University of Lübeck, 23562, Lübeck, Germany
| | - Fabian Ott
- Institute of Experimental Dermatology and Institute of Cardiogenetics, University of Lübeck, 23562, Lübeck, Germany
| | - Vladimir S Kostic
- Institute of Neurology, School of Medicine, University of Belgrade, Belgrade, 11000, Serbia
| | - Agata Gajos
- Department of Extrapyramidal Diseases, Medical University of Lodz, Lodz, 90-647, Poland
| | - Alexander Münchau
- Institute of Systems Motor Science, University of Lübeck, 23562, Lübeck, Germany
| | - Simone Zittel
- Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Hauke Busch
- Institute of Experimental Dermatology and Institute of Cardiogenetics, University of Lübeck, 23562, Lübeck, Germany
| | - Anne Grünewald
- Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, L-4362, Luxembourg
| | - Christine Klein
- Institute of Neurogenetics, University of Lübeck, 23562, Lübeck, Germany
| | - Katja Lohmann
- Institute of Neurogenetics, University of Lübeck, 23562, Lübeck, Germany.
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17
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Ko J, Song J, Choi N, Kim HN. Patient-Derived Microphysiological Systems for Precision Medicine. Adv Healthc Mater 2024; 13:e2303161. [PMID: 38010253 PMCID: PMC11469251 DOI: 10.1002/adhm.202303161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2023] [Indexed: 11/29/2023]
Abstract
Patient-derived microphysiological systems (P-MPS) have emerged as powerful tools in precision medicine that provide valuable insight into individual patient characteristics. This review discusses the development of P-MPS as an integration of patient-derived samples, including patient-derived cells, organoids, and induced pluripotent stem cells, into well-defined MPSs. Emphasizing the necessity of P-MPS development, its significance as a nonclinical assessment approach that bridges the gap between traditional in vitro models and clinical outcomes is highlighted. Additionally, guidance is provided for engineering approaches to develop microfluidic devices and high-content analysis for P-MPSs, enabling high biological relevance and high-throughput experimentation. The practical implications of the P-MPS are further examined by exploring the clinically relevant outcomes obtained from various types of patient-derived samples. The construction and analysis of these diverse samples within the P-MPS have resulted in physiologically relevant data, paving the way for the development of personalized treatment strategies. This study describes the significance of the P-MPS in precision medicine, as well as its unique capacity to offer valuable insights into individual patient characteristics.
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Affiliation(s)
- Jihoon Ko
- Department of BioNano TechnologyGachon UniversitySeongnam‐siGyeonggi‐do13120Republic of Korea
| | - Jiyoung Song
- Brain Science InstituteKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
| | - Nakwon Choi
- Brain Science InstituteKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Division of Bio‐Medical Science & TechnologyKIST SchoolSeoul02792Republic of Korea
- KU‐KIST Graduate School of Converging Science and TechnologyKorea UniversitySeoul02841Republic of Korea
| | - Hong Nam Kim
- Brain Science InstituteKorea Institute of Science and Technology (KIST)Seoul02792Republic of Korea
- Division of Bio‐Medical Science & TechnologyKIST SchoolSeoul02792Republic of Korea
- School of Mechanical EngineeringYonsei UniversitySeoul03722Republic of Korea
- Yonsei‐KIST Convergence Research InstituteYonsei UniversitySeoul03722Republic of Korea
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18
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Hasegawa M, Miki K, Kawamura T, Takei Sasozaki I, Higashiyama Y, Tsuchida M, Kashino K, Taira M, Ito E, Takeda M, Ishida H, Higo S, Sakata Y, Miyagawa S. Gene correction and overexpression of TNNI3 improve impaired relaxation in engineered heart tissue model of pediatric restrictive cardiomyopathy. Dev Growth Differ 2024; 66:119-132. [PMID: 38193576 PMCID: PMC11457505 DOI: 10.1111/dgd.12909] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2023] [Revised: 12/20/2023] [Accepted: 12/24/2023] [Indexed: 01/10/2024]
Abstract
Research on cardiomyopathy models using engineered heart tissue (EHT) created from disease-specific induced pluripotent stem cells (iPSCs) is advancing rapidly. However, the study of restrictive cardiomyopathy (RCM), a rare and intractable cardiomyopathy, remains at the experimental stage because there is currently no established method to replicate the hallmark phenotype of RCM, particularly diastolic dysfunction, in vitro. In this study, we generated iPSCs from a patient with early childhood-onset RCM harboring the TNNI3 R170W mutation (R170W-iPSCs). The properties of R170W-iPSC-derived cardiomyocytes (CMs) and EHTs were evaluated and compared with an isogenic iPSC line in which the mutation was corrected. Our results indicated altered calcium kinetics in R170W-iPSC-CMs, including prolonged tau, and an increased ratio of relaxation force to contractile force in R170W-EHTs. These properties were reversed in the isogenic line, suggesting that our model recapitulates impaired relaxation of RCM, i.e., diastolic dysfunction in clinical practice. Furthermore, overexpression of wild-type TNNI3 in R170W-iPSC-CMs and -EHTs effectively rescued impaired relaxation. These results highlight the potential efficacy of EHT, a modality that can accurately recapitulate diastolic dysfunction in vitro, to elucidate the pathophysiology of RCM, as well as the possible benefits of gene therapies for patients with RCM.
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Affiliation(s)
- Moyu Hasegawa
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Kenji Miki
- Premium Research Institute for Human Metaverse MedicineOsaka UniversityOsakaJapan
| | - Takuji Kawamura
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Ikue Takei Sasozaki
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Yuki Higashiyama
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Masaru Tsuchida
- NTT Communication Science LaboratoriesMedia Information Research DepartmentKanagawaJapan
| | - Kunio Kashino
- Premium Research Institute for Human Metaverse MedicineOsaka UniversityOsakaJapan
- NTT Communication Science LaboratoriesMedia Information Research DepartmentKanagawaJapan
| | - Masaki Taira
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Emiko Ito
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Maki Takeda
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
| | - Hidekazu Ishida
- Department of PediatricsOsaka University Graduate School of MedicineOsakaJapan
| | - Shuichiro Higo
- Department of Medical Therapeutics for Heart FailureOsaka University Graduate School of MedicineOsakaJapan
| | - Yasushi Sakata
- Department of Cardiovascular MedicineOsaka University Graduate School of MedicineOsakaJapan
| | - Shigeru Miyagawa
- Department of Cardiovascular SurgeryOsaka University Graduate School of MedicineOsakaJapan
- Premium Research Institute for Human Metaverse MedicineOsaka UniversityOsakaJapan
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19
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Dhanjal DS, Singh R, Sharma V, Nepovimova E, Adam V, Kuca K, Chopra C. Advances in Genetic Reprogramming: Prospects from Developmental Biology to Regenerative Medicine. Curr Med Chem 2024; 31:1646-1690. [PMID: 37138422 DOI: 10.2174/0929867330666230503144619] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 03/13/2023] [Accepted: 03/16/2023] [Indexed: 05/05/2023]
Abstract
The foundations of cell reprogramming were laid by Yamanaka and co-workers, who showed that somatic cells can be reprogrammed into pluripotent cells (induced pluripotency). Since this discovery, the field of regenerative medicine has seen advancements. For example, because they can differentiate into multiple cell types, pluripotent stem cells are considered vital components in regenerative medicine aimed at the functional restoration of damaged tissue. Despite years of research, both replacement and restoration of failed organs/ tissues have remained elusive scientific feats. However, with the inception of cell engineering and nuclear reprogramming, useful solutions have been identified to counter the need for compatible and sustainable organs. By combining the science underlying genetic engineering and nuclear reprogramming with regenerative medicine, scientists have engineered cells to make gene and stem cell therapies applicable and effective. These approaches have enabled the targeting of various pathways to reprogramme cells, i.e., make them behave in beneficial ways in a patient-specific manner. Technological advancements have clearly supported the concept and realization of regenerative medicine. Genetic engineering is used for tissue engineering and nuclear reprogramming and has led to advances in regenerative medicine. Targeted therapies and replacement of traumatized , damaged, or aged organs can be realized through genetic engineering. Furthermore, the success of these therapies has been validated through thousands of clinical trials. Scientists are currently evaluating induced tissue-specific stem cells (iTSCs), which may lead to tumour-free applications of pluripotency induction. In this review, we present state-of-the-art genetic engineering that has been used in regenerative medicine. We also focus on ways that genetic engineering and nuclear reprogramming have transformed regenerative medicine and have become unique therapeutic niches.
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Affiliation(s)
- Daljeet Singh Dhanjal
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
| | - Reena Singh
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
| | - Varun Sharma
- Head of Bioinformatic Division, NMC Genetics India Pvt. Ltd., Gurugram, India
| | - Eugenie Nepovimova
- Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, 50003, Czech Republic
| | - Vojtech Adam
- Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, Brno, CZ 613 00, Czech Republic
- Central European Institute of Technology, Brno University of Technology, Purkynova 123, Brno, CZ-612 00, Czech Republic
| | - Kamil Kuca
- Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, 50003, Czech Republic
- Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, 50005, Czech Republic
| | - Chirag Chopra
- School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
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20
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Chehelgerdi M, Behdarvand Dehkordi F, Chehelgerdi M, Kabiri H, Salehian-Dehkordi H, Abdolvand M, Salmanizadeh S, Rashidi M, Niazmand A, Ahmadi S, Feizbakhshan S, Kabiri S, Vatandoost N, Ranjbarnejad T. Exploring the promising potential of induced pluripotent stem cells in cancer research and therapy. Mol Cancer 2023; 22:189. [PMID: 38017433 PMCID: PMC10683363 DOI: 10.1186/s12943-023-01873-0] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Accepted: 09/27/2023] [Indexed: 11/30/2023] Open
Abstract
The advent of iPSCs has brought about a significant transformation in stem cell research, opening up promising avenues for advancing cancer treatment. The formation of cancer is a multifaceted process influenced by genetic, epigenetic, and environmental factors. iPSCs offer a distinctive platform for investigating the origin of cancer, paving the way for novel approaches to cancer treatment, drug testing, and tailored medical interventions. This review article will provide an overview of the science behind iPSCs, the current limitations and challenges in iPSC-based cancer therapy, the ethical and social implications, and the comparative analysis with other stem cell types for cancer treatment. The article will also discuss the applications of iPSCs in tumorigenesis, the future of iPSCs in tumorigenesis research, and highlight successful case studies utilizing iPSCs in tumorigenesis research. The conclusion will summarize the advancements made in iPSC-based tumorigenesis research and the importance of continued investment in iPSC research to unlock the full potential of these cells.
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Affiliation(s)
- Matin Chehelgerdi
- Novin Genome (NG) Lab, Research and Development Center for Biotechnology, Shahrekord, Iran
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | - Fereshteh Behdarvand Dehkordi
- Novin Genome (NG) Lab, Research and Development Center for Biotechnology, Shahrekord, Iran
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | - Mohammad Chehelgerdi
- Novin Genome (NG) Lab, Research and Development Center for Biotechnology, Shahrekord, Iran.
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran.
| | - Hamidreza Kabiri
- Novin Genome (NG) Lab, Research and Development Center for Biotechnology, Shahrekord, Iran
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | | | - Mohammad Abdolvand
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Sharareh Salmanizadeh
- Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Hezar-Jereeb Street, Isfahan, 81746-73441, Iran
| | - Mohsen Rashidi
- Department Pharmacology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
- The Health of Plant and Livestock Products Research Center, Mazandaran University of Medical Sciences, Sari, Iran
| | - Anoosha Niazmand
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Saba Ahmadi
- Department of Molecular and Medical Genetics, Tbilisi State Medical University, Tbilisi, Georgia
| | - Sara Feizbakhshan
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
| | - Saber Kabiri
- Novin Genome (NG) Lab, Research and Development Center for Biotechnology, Shahrekord, Iran
- Young Researchers and Elite Club, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | - Nasimeh Vatandoost
- Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan University of Medical Sciences, Isfahan, Iran
| | - Tayebeh Ranjbarnejad
- Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Science, Isfahan, Iran
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21
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Chen C, Tang X, Lan Z, Chen W, Su H, Li W, Li Y, Zhou X, Gao H, Feng X, Guo Y, Yao M, Deng W. GABAergic signaling abnormalities in a novel CLU mutation Alzheimer's disease mouse model. Transl Res 2023; 260:32-45. [PMID: 37211336 DOI: 10.1016/j.trsl.2023.05.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/04/2023] [Revised: 05/13/2023] [Accepted: 05/15/2023] [Indexed: 05/23/2023]
Abstract
The CLU rs11136000C mutation (CLUC) is the third most common risk factor for Alzheimer's disease (AD). However, the mechanism by which CLUC leads to abnormal GABAergic signaling in AD is unclear. To address this question, this study establishes the first chimeric mouse model of CLUC AD. Examination of grafted CLUC medial ganglionic eminence progenitors (CLUC hiMGEs) revealed increased GAD65/67 and a high frequency of spontaneous releasing events. CLUC hiMGEs also impaired cognition in chimeric mice and caused AD-related pathologies. The expression of GABA A receptor, subunit alpha 2 (Gabrα2) was higher in chimeric mice. Interestingly, cognitive impairment in chimeric mice was reversed by treatment with pentylenetetrazole, which is a GABA A receptor inhibitor. Taken together, these findings shed light on the pathogenesis of CLUC AD using a novel humanized animal model and suggest sphingolipid signaling over-activation as a potential mechanism of GABAergic signaling disorder.
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Affiliation(s)
- Chunxia Chen
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China; Department of pharmacy, The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, Guangxi P. R. China
| | - Xihe Tang
- Department of neurosurgery, The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, Guangxi P. R. China; Department of neurosurgery, Aviation General Hospital, Beijing, P. R. China
| | - Zhaohui Lan
- Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education), Shanghai Key Laboratory of Psychotic Disorders, and Brain Science and Technology Research Center, Bio-X Institutes, Institute of Psychology and Behavioral Sciences, Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Wan Chen
- Department of Emergency, The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, Guangxi, P. R. China
| | - Hua Su
- Department of Pharmacology, Guangxi Institute of Chinese Medicine & Pharmaceutical Science, Nanning, P. R. China
| | - Weidong Li
- Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education), Shanghai Key Laboratory of Psychotic Disorders, and Brain Science and Technology Research Center, Bio-X Institutes, Institute of Psychology and Behavioral Sciences, Shanghai Jiao Tong University, Shanghai, P. R. China
| | - Yaoxuan Li
- Department of Neurology, The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, Guangxi, P. R. China
| | - Xing Zhou
- Department of pharmacy, The People's Hospital of Guangxi Zhuang Autonomous Region & Guangxi Academy of Medical Sciences, Nanning, Guangxi P. R. China
| | - Hong Gao
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China
| | - Xinwei Feng
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China
| | - Ying Guo
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China
| | - Meicun Yao
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China
| | - Wenbin Deng
- School of Pharmaceutical Sciences, Shenzhen Campus of Sun Yat-sen University, Shenzhen, Guangdong P. R. China.
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22
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Kistamás K, Müller A, Muenthaisong S, Lamberto F, Zana M, Dulac M, Leal F, Maziz A, Costa P, Bernotiene E, Bergaud C, Dinnyés A. Multifactorial approaches to enhance maturation of human iPSC-derived cardiomyocytes. J Mol Liq 2023; 387:122668. [DOI: 10.1016/j.molliq.2023.122668] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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23
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Izadifar M, Berecz T, Li B, Tang JKKY, Foldes G, Apati A, Nagy A. Speckle-Tracking Strain Analysis for Mapping Spatiotemporal Contractility of Induced Pluripotent Stem Cell (iPSC)-Derived Cardiomyocytes. Curr Protoc 2023; 3:e889. [PMID: 37747346 DOI: 10.1002/cpz1.889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/26/2023]
Abstract
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) hold tremendous potential for cardiovascular disease modeling, drug screening, personalized medicine, and pathophysiology studies. The availability of a robust protocol and functional assay for studying phenotypic behavior of hiPSC-CMs is essential for establishing an in vitro disease model. Many heart diseases manifest due to changes in the mechanical strain of cardiac tissue. Therefore, non-invasive evaluation of the contractility properties of hiPSC-CMs remains crucial to gain an insight into the pathogenesis of cardiac diseases. Speckle tracking-based strain analysis is an efficient non-invasive method that uses video microscopy and image analysis of beating hiPSC-CMs for quantitative evaluation of mechanical contractility properties. This article presents step-by-step protocols for extracting quantitative contractility properties of an hiPSC-CM system obtained from five members of a family, of whom three were affected by DiGeorge syndrome, using speckle tracking-based strain analysis. The hiPSCs from the family members were differentiated and purified into hiPSC-CMs using metabolic selection. Time-lapse images of hiPSC-CMs were acquired using high-spatial-resolution and high-time-resolution phase-contrast video microscopy. Speckled images were characterized by evaluating the cross-correlation coefficient, speckle size, speckle contrast, and speckle quality of the images. The optimum parameters of the speckle tracking algorithm were determined by performing sensitivity analysis concerning computation time, effective mapping area, average contraction velocity, and strain. Furthermore, the hiPSC-CM response to adrenaline was evaluated to validate the sensitivity of the strain analysis algorithm. Then, we applied speckle tracking-based strain analysis to characterize the dynamic behavior of patient-specific hiPSC-CMs from the family members affected/unaffected by DiGeorge syndrome. Here, we report an efficient and manipulation-free method to analyze the contraction displacement vector and velocity field, contraction-relaxation strain rate, and contractile cycles. Implementation of this method allows for quantitative analysis of the contractile phenotype characteristics of hiPSC-CMs to distinguish possible cardiac manifestation of DiGeorge syndrome. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of iPSCs into iPSC-derived cardiomyocytes (iPSC-CMs) and metabolic selection of differentiated iPSC-CMs Support Protocol 1: Culture, maintenance, and expansion of human iPSCs Support Protocol 2: Immunohistochemistry of iPSC-CMs Basic Protocol 2: Time-lapse speckle imaging of iPSC-CMs and speckle quality characterization Support Protocol 3: Enhancement of local contrast of videos by applying contrast limited adaptive histogram equalization (CLAHE) to all frames Support Protocol 4: Evaluation of average speckle size Support Protocol 5: Evaluation of average speckle contrast Support Protocol 6: Determination of relative peak height, Pc(x), of consecutive images acquired from video microscopy of iPSC-CMs Basic Protocol 3: Speckle tracking-based analysis of beating iPSC-CMs Support Protocol 7: Validation of sensitivity of the speckle tracking analysis for mapping the contractility of iPSC-CMs Basic Protocol 4: Data extraction, visualization, and mapping of contractile cycles of iPSC-CMs.
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Affiliation(s)
- Mohammad Izadifar
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Tunde Berecz
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
| | - Biao Li
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | | | - Gabor Foldes
- Heart and Vascular Center, Semmelweis University, Budapest, Hungary
| | - Agota Apati
- Institute of Enzymology, Research Centre for Natural Sciences, Budapest, Hungary
| | - Andras Nagy
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia
- Department of Obstetrics and Gynecology, University of Toronto, Toronto, Ontario, Canada
- Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
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24
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Milagre I, Pereira C, Oliveira RA. Compromised Mitotic Fidelity in Human Pluripotent Stem Cells. Int J Mol Sci 2023; 24:11933. [PMID: 37569309 PMCID: PMC10418648 DOI: 10.3390/ijms241511933] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2023] [Revised: 07/19/2023] [Accepted: 07/21/2023] [Indexed: 08/13/2023] Open
Abstract
Human pluripotent stem cells (PSCs), which include both embryonic and induced pluripotent stem cells, are widely used in fundamental and applied biomedical research. They have been instrumental for better understanding development and cell differentiation processes, disease origin and progression and can aid in the discovery of new drugs. PSCs also hold great potential in regenerative medicine to treat or diminish the effects of certain debilitating diseases, such as degenerative disorders. However, some concerns have recently been raised over their safety for use in regenerative medicine. One of the major concerns is the fact that PSCs are prone to errors in passing the correct number of chromosomes to daughter cells, resulting in aneuploid cells. Aneuploidy, characterised by an imbalance in chromosome number, elicits the upregulation of different stress pathways that are deleterious to cell homeostasis, impair proper embryo development and potentiate cancer development. In this review, we will summarize known molecular mechanisms recently revealed to impair mitotic fidelity in human PSCs and the consequences of the decreased mitotic fidelity of these cells. We will finish with speculative views on how the physiological characteristics of PSCs can affect the mitotic machinery and how their suboptimal mitotic fidelity may be circumvented.
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Affiliation(s)
- Inês Milagre
- Católica Biomedical Research Centre, Católica Medical School, Universidade Católica Portuguesa, 1649-023 Lisbon, Portugal
| | | | - Raquel A. Oliveira
- Católica Biomedical Research Centre, Católica Medical School, Universidade Católica Portuguesa, 1649-023 Lisbon, Portugal
- Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal
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25
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Fujita H, Kaneshiro J, Takeda M, Sasaki K, Yamamoto R, Umetsu D, Kuranaga E, Higo S, Kondo T, Asano Y, Sakata Y, Miyagawa S, Watanabe TM. Estimation of crossbridge-state during cardiomyocyte beating using second harmonic generation. Life Sci Alliance 2023; 6:e202302070. [PMID: 37236659 PMCID: PMC10215972 DOI: 10.26508/lsa.202302070] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2023] [Revised: 04/21/2023] [Accepted: 04/21/2023] [Indexed: 05/28/2023] Open
Abstract
Estimation of dynamic change of crossbridge formation in living cardiomyocytes is expected to provide crucial information for elucidating cardiomyopathy mechanisms, efficacy of an intervention, and others. Here, we established an assay system to dynamically measure second harmonic generation (SHG) anisotropy derived from myosin filaments depended on their crossbridge status in pulsating cardiomyocytes. Experiments utilizing an inheritable mutation that induces excessive myosin-actin interactions revealed that the correlation between sarcomere length and SHG anisotropy represents crossbridge formation ratio during pulsation. Furthermore, the present method found that ultraviolet irradiation induced an increased population of attached crossbridges that lost the force-generating ability upon myocardial differentiation. Taking an advantage of infrared two-photon excitation in SHG microscopy, myocardial dysfunction could be intravitally evaluated in a Drosophila disease model. Thus, we successfully demonstrated the applicability and effectiveness of the present method to evaluate the actomyosin activity of a drug or genetic defect on cardiomyocytes. Because genomic inspection alone may not catch the risk of cardiomyopathy in some cases, our study demonstrated herein would be of help in the risk assessment of future heart failure.
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Affiliation(s)
- Hideaki Fujita
- Department of Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Junichi Kaneshiro
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Maki Takeda
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Kensuke Sasaki
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
| | - Rikako Yamamoto
- Department of Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
| | - Daiki Umetsu
- Laboratory for Histogenetic Dynamics, Graduate School of Life Sciences, Tohoku University, Sendai, Japan
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan
| | - Erina Kuranaga
- Laboratory for Histogenetic Dynamics, Graduate School of Life Sciences, Tohoku University, Sendai, Japan
| | - Shuichiro Higo
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Takumi Kondo
- Department of Medical Therapeutics for Heart Failure, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Yoshihiro Asano
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Yasushi Sakata
- Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan
| | - Tomonobu M Watanabe
- Department of Stem Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan
- Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
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26
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Plakkot B, Di Agostino A, Subramanian M. Implications of Hypothalamic Neural Stem Cells on Aging and Obesity-Associated Cardiovascular Diseases. Cells 2023; 12:cells12050769. [PMID: 36899905 PMCID: PMC10000584 DOI: 10.3390/cells12050769] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 02/14/2023] [Accepted: 02/25/2023] [Indexed: 03/06/2023] Open
Abstract
The hypothalamus, one of the major regulatory centers in the brain, controls various homeostatic processes, and hypothalamic neural stem cells (htNSCs) have been observed to interfere with hypothalamic mechanisms regulating aging. NSCs play a pivotal role in the repair and regeneration of brain cells during neurodegenerative diseases and rejuvenate the brain tissue microenvironment. The hypothalamus was recently observed to be involved in neuroinflammation mediated by cellular senescence. Cellular senescence, or systemic aging, is characterized by a progressive irreversible state of cell cycle arrest that causes physiological dysregulation in the body and it is evident in many neuroinflammatory conditions, including obesity. Upregulation of neuroinflammation and oxidative stress due to senescence has the potential to alter the functioning of NSCs. Various studies have substantiated the chances of obesity inducing accelerated aging. Therefore, it is essential to explore the potential effects of htNSC dysregulation in obesity and underlying pathways to develop strategies to address obesity-induced comorbidities associated with brain aging. This review will summarize hypothalamic neurogenesis associated with obesity and prospective NSC-based regenerative therapy for the treatment of obesity-induced cardiovascular conditions.
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27
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Spinal cord extracts of amyotrophic lateral sclerosis spread TDP-43 pathology in cerebral organoids. PLoS Genet 2023; 19:e1010606. [PMID: 36745687 PMCID: PMC9934440 DOI: 10.1371/journal.pgen.1010606] [Citation(s) in RCA: 28] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 02/16/2023] [Accepted: 01/09/2023] [Indexed: 02/07/2023] Open
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.
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28
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Portela-Lomba M, Simón D, Fernández de Sevilla D, Moreno-Flores MT, Sierra J. Small molecules fail to induce direct reprogramming of adult rat olfactory ensheathing glia to mature neurons. Front Mol Neurosci 2023; 16:1110356. [PMID: 36910262 PMCID: PMC9998535 DOI: 10.3389/fnmol.2023.1110356] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2022] [Accepted: 01/30/2023] [Indexed: 03/14/2023] Open
Abstract
An approach to generate new neurons after central nervous system injury or disease is direct reprogramming of the individual's own somatic cells into differentiated neurons. This can be achieved either by transduction of viral vectors that express neurogenic transcription factors and/or through induction with small molecules, avoiding introducing foreign genetic material in target cells. In this work, we propose olfactory ensheathing glia (OEG) as a candidate for direct reprogramming to neurons with small molecules due to its well-characterized neuro-regenerative capacity. After screening different combinations of small molecules in different culture conditions, only partial reprogramming was achieved: induced cells expressed neuronal markers but lacked the ability of firing action potentials. Our work demonstrates that direct conversion of adult olfactory ensheathing glia to mature, functional neurons cannot be induced only with pharmacological tools.
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Affiliation(s)
- María Portela-Lomba
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain
| | - Diana Simón
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain
| | - David Fernández de Sevilla
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - Mª Teresa Moreno-Flores
- Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain
| | - Javier Sierra
- School of Experimental Sciences, Universidad Francisco de Vitoria, Pozuelo de Alarcón, Madrid, Spain.,School of Medicine, Universidad Francisco de Vitoria, Madrid, Spain
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29
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Long-term calcium imaging reveals functional development in hiPSC-derived cultures comparable to human but not rat primary cultures. Stem Cell Reports 2022; 18:205-219. [PMID: 36563684 PMCID: PMC9860124 DOI: 10.1016/j.stemcr.2022.11.014] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2022] [Revised: 11/15/2022] [Accepted: 11/18/2022] [Indexed: 12/24/2022] Open
Abstract
Models for human brain-oriented research are often established on primary cultures from rodents, which fails to recapitulate cellular specificity and molecular cues of the human brain. Here we investigated whether neuronal cultures derived from human induced pluripotent stem cells (hiPSCs) feature key advantages compared with rodent primary cultures. Using calcium fluorescence imaging, we tracked spontaneous neuronal activity in hiPSC-derived, human, and rat primary cultures and compared their dynamic and functional behavior as they matured. We observed that hiPSC-derived cultures progressively changed upon development, exhibiting gradually richer activity patterns and functional traits. By contrast, rat primary cultures were locked in the same dynamic state since activity onset. Human primary cultures exhibited features in between hiPSC-derived and rat primary cultures, although traits from the former predominated. Our study demonstrates that hiPSC-derived cultures are excellent models to investigate development in neuronal assemblies, a hallmark for applications that monitor alterations caused by damage or neurodegeneration.
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30
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Azeez IA, Awogbindin IO, Olayinka JN, Folarin RO, Adamu AS, Ior LD, Shehu AM, Mukhtar AI, Ajeigbe OF, Emokpae AO, Usende IL, Babatunde BR, Yusha'u Y, Olateju OI, Kamoga R, Benson AIO, Oparaji KC, Owemidu IO, Iliyasu MO, Imam MI, Olopade JO. Neural stem cell research in Africa: current realities and future prospects. Biol Open 2022; 11:280534. [PMID: 36326097 PMCID: PMC9641530 DOI: 10.1242/bio.059574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Neural stem cells (NSCs) are immature progenitor cells that are found in developing and adult brains that have the potential of dividing actively and renewing themselves, with a complex form of gene expression. The generation of new brain cells in adult individuals was initially considered impossible, however, the landmark discovery of human neural stem cells in the hippocampus has been followed by further discoveries in other discreet regions of the brain. Investigation into the current state in Africa of the research and use of NSCs shows relatively limited activities on the continent. Information on the African application of NSCs for modelling disease mechanisms, drug discovery, and therapeutics is still limited. The International Brain Research Organization (IBRO)-African Regional Committee (ARC), with support from the Company of Biologists, and the Movement Disorder Society, sponsored the first African Basic School on NSC in Ibadan, Nigeria, with the vision of bringing together young neuroscientists and physicians across different fields in neuroscience to learn from leaders who have applied NSCs in stem cell research, the pathophysiology of neurodegenerative diseases, neuroanatomy, and neurotherapeutics. Twenty early-career researchers in academic institutions at junior and senior faculty cadres were selected from South Africa, Uganda and Nigeria. The students and organizer of the school, who wrote this review on the state of NSCs research in Africa, recommended the following: (1) other African countries can take a cue from South Africa and Nigeria in probing the phenomena of adult neurogenesis in unique animal species on the continent; (2) Africa should leverage the expertise and facilities of South African scientists and international collaborators in scaling up NSC research into these unique species and (3) Centers of Excellence should be established on the continent to serve as research hubs for training postgraduate students, and facilities for African scientists who trained overseas on NSCs.
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Affiliation(s)
- Idris A. Azeez
- Department of Veterinary Anatomy, University of Jos 1 , Jos, 930001 Nigeria
| | | | - Juliet N. Olayinka
- Department of Pharmacology and Therapeutics, Afe Babalola University 3 , Ado-Ekiti, 360001 Nigeria
| | - Royhaan O. Folarin
- Department of Anatomy, Olabisi Onabanjo University 4 , Ago-Iwoye, 120107 Nigeria
| | - Abubakar S. Adamu
- Department of Human Anatomy, Ahmadu Bello University 5 , Zaria, 810107 , Nigeria
| | - Lydia D. Ior
- Department of Pharmacology, University of Jos 6 , Jos, 930001 , Nigeria
| | - Asmau M. Shehu
- Department of Human Anatomy, Federal University Dutse 7 , Dutse, 720223 , Nigeria
- School of Anatomical Sciences, University of the Witwatersrand 8 , Johannesburg, Wits 2050 , South Africa
| | - Abubakar I. Mukhtar
- Department of Human Anatomy, Ahmadu Bello University 5 , Zaria, 810107 , Nigeria
| | - Olufunke F. Ajeigbe
- Elizade University, Ilara-Mokin, 340112 9 Department of Physical and Chemical Sciences, Biochemistry Programme , , Nigeria
| | | | - Ifukibot L. Usende
- Department of Veterinary Anatomy, University of Abuja 11 , Abuja, 900105 , Nigeria
| | | | - Yusuf Yusha'u
- Department of Human Physiology, Ahmadu Bello University 12 , Zaria, 810107 , Nigeria
| | - Oladiran I. Olateju
- School of Anatomical Sciences, University of the Witwatersrand 8 , Johannesburg, Wits 2050 , South Africa
| | - Ronald Kamoga
- Department of Pharmacology and Therapeutics, Mbarara University of Science and Technology 13 , Mbarara P.O. Box 1410 , Uganda
| | - Ayoola I. O. Benson
- Department of Human Anatomy, Elizade University, Ilara-Mokin 14 , Abakaliki, 482131 Nigeria
| | - Kenneth C. Oparaji
- Department of Physiology, Alex Ekwueme Federal University Ndufu-Alike 15 , Abakaliki, 482131 , Nigeria
| | - Idowu O. Owemidu
- Department of Physiology, Kogi State University 16 , Anyigba, 272102 , Nigeria
| | - Musa O. Iliyasu
- Department of Anatomy, Kogi State University 17 , Anyigba, 272102 , Nigeria
| | - Maryam I. Imam
- Department of Human Physiology, Ahmadu Bello University 12 , Zaria, 810107 , Nigeria
| | - James O. Olopade
- Department of Veterinary Anatomy, University of Ibadan 18 , Ibadan, 200005 , Nigeria
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Snelders M, Koedijk IH, Schirmer J, Mulleners O, van Leeuwen J, de Wagenaar NP, Bartulos O, Voskamp P, Braam S, Guttenberg Z, Danser AJ, Majoor-Krakauer D, Meijering E, van der Pluijm I, Essers J. Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness. BIOMATERIALS AND BIOSYSTEMS 2022; 8:100068. [PMID: 36824378 PMCID: PMC9934435 DOI: 10.1016/j.bbiosy.2022.100068] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 09/09/2022] [Accepted: 10/15/2022] [Indexed: 12/04/2022] Open
Abstract
Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with β-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. β-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.
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Affiliation(s)
- Matthijs Snelders
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands
| | - Iris H. Koedijk
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands
| | | | - Otto Mulleners
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands
| | | | - Nathalie P. de Wagenaar
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands,Department of Cardiology, Erasmus MC, Rotterdam, the Netherlands
| | | | | | | | | | - A.H. Jan Danser
- Department of Internal Medicine - Pharmacology, Erasmus MC, Rotterdam, the Netherlands
| | | | - Erik Meijering
- School of Computer Science and Engineering, University of New South Wales, Sydney, Australia
| | - Ingrid van der Pluijm
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands,Department of Vascular Surgery, Erasmus MC, Rotterdam, the Netherlands
| | - Jeroen Essers
- Department of Molecular Genetics, Erasmus MC, Rotterdam, the Netherlands,Department of Vascular Surgery, Erasmus MC, Rotterdam, the Netherlands,Department of Radiotherapy, Erasmus MC, Rotterdam, the Netherlands,Corresponding author: Erasmus Medical Center, Wytemaweg 80, Rotterdam 3015CN, The Netherlands
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Valle ACV, Brunel HDSS, Dallago BSL, Rodrigues LS, Malard PF, da Costa RA, Rossetto R, de Andrade RV. In-Vitro Growth Kinetics of Mesenchymal Stem Cells in Cytotoxicity Tests Using Low-Diluted Viscum Album. HOMEOPATHY 2022; 112:40-49. [PMID: 35988582 PMCID: PMC9868971 DOI: 10.1055/s-0042-1747682] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
INTRODUCTION The use of mesenchymal stem cells (MSC) in cytotoxicity tests is an in-vitro alternative model for predicting initial doses. Homeopathic medicines may stimulate the immune system to combat a pathology effectively and have been used for over two centuries. Viscum album (VA) extracts are widely used in the treatment of cancer, due to their immunomodulatory, cytotoxic and pro-apoptotic properties. OBJECTIVE This study aimed to evaluate the in-vitro growth kinetics of canine MSC in relation to cytotoxicity, cell differentiation and expression of pluripotentiality markers, using a VA preparation at the D1D2 (1×10-1, 1×10-2 potency (VAD1D2). METHODS MSC were obtained from adipose tissue sampled from a healthy dog that was undergoing an elective veterinary procedure and with its owner's permission. The experiments were performed in three groups: MSC treated with VAD1D2 or diluent or untreated (control). The cytotoxicity was evaluated by MTT assay. The differentiation was induced in three lineages, and apoptotic cell labeling was performed by an Annexin-V test. RESULTS At the concentration of 10 μL/mL of VA, the number of cells after in-vitro culture was maintained when compared with the control (untreated) group. A significant and gradual decrease in cell viability was recorded as VA concentrations increased. The apoptosis analysis showed that VA at 20 μL/mL presented absolute percentages of initial apoptosis twice as high as at 10 μL/mL, which was similar to the control (untreated group). CONCLUSION The results suggest that the use of efficient methods to assess the in-vitro cytotoxicity of VA-based homeopathic medicines using MSC lineages may predict the potential action at different concentrations. These findings demonstrated that VAD1D2 interferes with canine MSC growth kinetics.
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Affiliation(s)
- Ana Catarina Viana Valle
- Doctor Izao Soares Institute, Sao Paulo, Brazil,Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
| | | | | | | | - Patrícia Furtado Malard
- Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil,BioCell Cell Therapy, Brazil
| | | | - Rafael Rossetto
- Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil
| | - Rosângela Vieira de Andrade
- Graduate Program in Genomic Sciences and Biotechnology, Catholic University of Brasilia, Brazil,Address for correspondence Rosangela Vieira de Andrade, PhD GGraduate Program in Genomic Sciences and Biotechnology, Catholic University of BrasiliaSGAN 916, Brasília, DF 70790-160Brazil
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Zhang L, Qin Z, Lyu D, Lu B, Chen Z, Fu Q, Yao K. Postponement of the opacification of lentoid bodies derived from human induced pluripotent stem cells after lanosterol treatment-the first use of the lens aging model in vitro in cataract drug screening. Front Pharmacol 2022; 13:959978. [PMID: 36059984 PMCID: PMC9437520 DOI: 10.3389/fphar.2022.959978] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Accepted: 07/18/2022] [Indexed: 11/17/2022] Open
Abstract
Purpose: Our previous study observed that human induced pluripotent stem cell (HiPSC)-derived lentoid bodies (LBs) became cloudy with extended culture time, partially mimicking the progress of human age-related cataracts (ARCs) in a dish. In the present study, lanosterol, a potential anticataract drug, was used to further verify the value of this model in drug screening for cataract treatment. Methods: Mature LBs on day 25, which were differentiated from HiPSCs using the "fried egg" method, were continually cultured and treated with either dimethyl sulfoxide (control) or lanosterol. The LBs' shape and opacity alterations were examined using light microscopy and mean gray value evaluation. The soluble and insoluble proteins were examined through SDS-PAGE gel electrophoresis combined with Coomassie blue staining. The protein aggregations were examined with immunofluorescence. Results: The mature LBs became cloudy with an extended culture time, and the opacification of the LBs was partially prevented by lanosterol treatment. There was less increase in insoluble proteins in the lanosterol-treated LBs than in the control group. There were also fewer cells containing aggregated protein (αA-crystallin and αB-crystallin) puncta in the lanosterol-treated LBs than in the control LBs. Conclusion: It was found that the opacification of LBs could be delayed by lanosterol treatment, which could be achieved by reducing protein aggregation, suggesting a promising HiPSC-derived drug-screening model for Age-related cataract.
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Affiliation(s)
- Lifang Zhang
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
- Department of Ophthalmology, The Affiliated People’s Hospital of Ningbo University, Ningbo, China
| | - Zhenwei Qin
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
| | - Danni Lyu
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
| | - Bing Lu
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
| | - Zhijian Chen
- Department of Environmental and Occupational Health, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China
| | - Qiuli Fu
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
| | - Ke Yao
- Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China
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Inagaki E, Yoshimatsu S, Okano H. Accelerated neuronal aging in vitro ∼melting watch ∼. Front Aging Neurosci 2022; 14:868770. [PMID: 36016855 PMCID: PMC9397486 DOI: 10.3389/fnagi.2022.868770] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 07/04/2022] [Indexed: 11/13/2022] Open
Abstract
In developed countries, the aging of the population and the associated increase in age-related diseases are causing major unresolved medical, social, and environmental matters. Therefore, research on aging has become one of the most important and urgent issues in life sciences. If the molecular mechanisms of the onset and progression of neurodegenerative diseases are elucidated, we can expect to develop disease-modifying methods to prevent neurodegeneration itself. Since the discovery of induced pluripotent stem cells (iPSCs), there has been an explosion of disease models using disease-specific iPSCs derived from patient-derived somatic cells. By inducing the differentiation of iPSCs into neurons, disease models that reflect the patient-derived pathology can be reproduced in culture dishes, and are playing an active role in elucidating new pathological mechanisms and as a platform for new drug discovery. At the same time, however, we are faced with a new problem: how to recapitulate aging in culture dishes. It has been pointed out that cells differentiated from pluripotent stem cells are juvenile, retain embryonic traits, and may not be fully mature. Therefore, attempts are being made to induce cell maturation, senescence, and stress signals through culture conditions. It has also been reported that direct conversion of fibroblasts into neurons can reproduce human neurons with an aged phenotype. Here, we outline some state-of-the-art insights into models of neuronal aging in vitro. New frontiers in which stem cells and methods for inducing differentiation of tissue regeneration can be applied to aging research are just now approaching, and we need to keep a close eye on them. These models are forefront and intended to advance our knowledge of the molecular mechanisms of aging and contribute to the development of novel therapies for human neurodegenerative diseases associated with aging.
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Affiliation(s)
- Emi Inagaki
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
- Japanese Society for the Promotion of Science (JSPS), Tokyo, Japan
| | - Sho Yoshimatsu
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
- Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
| | - Hideyuki Okano
- Department of Physiology, Keio University School of Medicine, Tokyo, Japan
- *Correspondence: Hideyuki Okano,
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Zhang SY, Zhao J, Ni JJ, Li H, Quan ZZ, Qing H. Application and prospects of high-throughput screening for in vitro neurogenesis. World J Stem Cells 2022; 14:393-419. [PMID: 35949394 PMCID: PMC9244953 DOI: 10.4252/wjsc.v14.i6.393] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2021] [Revised: 04/07/2022] [Accepted: 05/28/2022] [Indexed: 02/06/2023] Open
Abstract
Over the past few decades, high-throughput screening (HTS) has made great contributions to new drug discovery. HTS technology is equipped with higher throughput, minimized platforms, more automated and computerized operating systems, more efficient and sensitive detection devices, and rapid data processing systems. At the same time, in vitro neurogenesis is gradually becoming important in establishing models to investigate the mechanisms of neural disease or developmental processes. However, challenges remain in generating more mature and functional neurons with specific subtypes and in establishing robust and standardized three-dimensional (3D) in vitro models with neural cells cultured in 3D matrices or organoids representing specific brain regions. Here, we review the applications of HTS technologies on in vitro neurogenesis, especially aiming at identifying the essential genes, chemical small molecules and adaptive microenvironments that hold great prospects for generating functional neurons or more reproductive and homogeneous 3D organoids. We also discuss the developmental tendency of HTS technology, e.g., so-called next-generation screening, which utilizes 3D organoid-based screening combined with microfluidic devices to narrow the gap between in vitro models and in vivo situations both physiologically and pathologically.
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Affiliation(s)
- Shu-Yuan Zhang
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Juan Zhao
- Aerospace Medical Center, Aerospace Center Hospital, Beijing 100049, China
| | - Jun-Jun Ni
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Hui Li
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Zhen-Zhen Quan
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
| | - Hong Qing
- Key Laboratory of Molecular Medicine and Biotherapy in the Ministry of Industry and Information Technology, Department of Biology, School of Life Science, Beijing Institute of Technology, Beijing 100081, China
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Doi M, Li M, Usui N, Shimada S. Genomic Strategies for Understanding the Pathophysiology of Autism Spectrum Disorder. Front Mol Neurosci 2022; 15:930941. [PMID: 35813066 PMCID: PMC9263364 DOI: 10.3389/fnmol.2022.930941] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 06/06/2022] [Indexed: 11/25/2022] Open
Abstract
Recent breakthroughs in sequencing technology and technological developments have made it easier to analyze the entire human genome than ever before. In addition to disease-specific genetic mutations and chromosomal aberrations, epigenetic alterations in individuals can also be analyzed using genomics. Autism spectrum disorder (ASD) is a neurodevelopmental disorder (NDD) caused by genetic and/or environmental factors. More than a thousand genes associated with ASD have been identified which are known to be involved in brain development. However, it is difficult to decode the roles of ASD-associated genes without in vitro and in vivo validations, particularly in the process of brain development. In this review, we discuss genomic strategies for understanding the pathological mechanisms underlying ASD. For this purpose, we discuss ASD-associated genes and their functions, as well as analytical strategies and their strengths and weaknesses in cellular and animal models from a basic research perspective.
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Affiliation(s)
- Miyuki Doi
- Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Japan
- Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, Osaka, Japan
| | - Mengwei Li
- Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Japan
| | - Noriyoshi Usui
- Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Japan
- Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, Osaka, Japan
- United Graduate School of Child Development, Osaka University, Suita, Japan
- Global Center for Medical Engineering and Informatics, Osaka University, Suita, Japan
- *Correspondence: Noriyoshi Usui
| | - Shoichi Shimada
- Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, Suita, Japan
- Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, Osaka, Japan
- United Graduate School of Child Development, Osaka University, Suita, Japan
- Global Center for Medical Engineering and Informatics, Osaka University, Suita, Japan
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Rosholm KR, Badone B, Karatsiompani S, Nagy D, Seibertz F, Voigt N, Bell DC. Adventures and Advances in Time Travel With Induced Pluripotent Stem Cells and Automated Patch Clamp. Front Mol Neurosci 2022; 15:898717. [PMID: 35813069 PMCID: PMC9258620 DOI: 10.3389/fnmol.2022.898717] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Accepted: 05/13/2022] [Indexed: 01/21/2023] Open
Abstract
In the Hollywood blockbuster “The Curious Case of Benjamin Button” a fantastical fable unfolds of a man’s life that travels through time reversing the aging process; as the tale progresses, the frail old man becomes a vigorous, vivacious young man, then man becomes boy and boy becomes baby. The reality of cellular time travel, however, is far more wondrous: we now have the ability to both reverse and then forward time on mature cells. Four proteins were found to rewind the molecular clock of adult cells back to their embryonic, “blank canvas” pluripotent stem cell state, allowing these pluripotent stem cells to then be differentiated to fast forward their molecular clocks to the desired adult specialist cell types. These four proteins – the “Yamanaka factors” – form critical elements of this cellular time travel, which deservedly won Shinya Yamanaka the Nobel Prize for his lab’s work discovering them. Human induced pluripotent stem cells (hiPSCs) hold much promise in our understanding of physiology and medicine. They encapsulate the signaling pathways of the desired cell types, such as cardiomyocytes or neurons, and thus act as model cells for defining the critical ion channel activity in healthy and disease states. Since hiPSCs can be derived from any patient, highly specific, personalized (or stratified) physiology, and/or pathophysiology can be defined, leading to exciting developments in personalized medicines and interventions. As such, hiPSC married with high throughput automated patch clamp (APC) ion channel recording platforms provide a foundation for significant physiological, medical and drug discovery advances. This review aims to summarize the current state of affairs of hiPSC and APC: the background and recent advances made; and the pros, cons and challenges of these technologies. Whilst the authors have yet to finalize a fully functional time traveling machine, they will endeavor to provide plausible future projections on where hiPSC and APC are likely to carry us. One future projection the authors are confident in making is the increasing necessity and adoption of these technologies in the discovery of the next blockbuster, this time a life-enhancing ion channel drug, not a fantastical movie.
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Affiliation(s)
- Kadla R. Rosholm
- Sophion Bioscience A/S, Ballerup, Denmark
- *Correspondence: Kadla R. Rosholm,
| | | | | | - David Nagy
- Sophion Bioscience Inc., Woburn, MA, United States
| | - Fitzwilliam Seibertz
- Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany
- German Center for Cardiovascular Research (DZHK), Göttingen, Germany
| | - Niels Voigt
- Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany
- German Center for Cardiovascular Research (DZHK), Göttingen, Germany
- Cluster of Excellence “Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells” (MBExC), University of Göttingen, Göttingen, Germany
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Exploring the Potential of Symmetric Exon Deletion to Treat Non-Ischemic Dilated Cardiomyopathy by Removing Frameshift Mutations in TTN. Genes (Basel) 2022; 13:genes13061093. [PMID: 35741855 PMCID: PMC9222585 DOI: 10.3390/genes13061093] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 05/30/2022] [Accepted: 06/10/2022] [Indexed: 11/17/2022] Open
Abstract
Non-ischemic dilated cardiomyopathy (DCM) is one of the most frequent pathologies requiring cardiac transplants. Even though the etiology of this disease is complex, frameshift mutations in the giant sarcomeric protein Titin could explain up to 25% of the familial and 18% of the sporadic cases of DCM. Many studies have shown the potential of genome editing using CRISPR/Cas9 to correct truncating mutations in sarcomeric proteins and have established the grounds for myoediting. However, these therapies are still in an immature state, with only few studies showing an efficient treatment of cardiac diseases. This publication hypothesizes that the Titin (TTN)-specific gene structure allows the application of myoediting approaches in a broad range of locations to reframe TTNtvvariants and to treat DCM patients. Additionally, to pave the way for the generation of efficient myoediting approaches for DCM, we screened and selected promising target locations in TTN. We conceptually explored the deletion of symmetric exons as a therapeutic approach to restore TTN’s reading frame in cases of frameshift mutations. We identified a set of 94 potential candidate exons of TTN that we consider particularly suitable for this therapeutic deletion. With this study, we aim to contribute to the development of new therapies to efficiently treat titinopathies and other diseases caused by mutations in genes encoding proteins with modular structures, e.g., Obscurin.
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Hossain MA, Hasegawa-Ogawa M, Manome Y, Igarashi M, Wu C, Suzuki K, Igarashi J, Iwamoto T, Okano HJ, Eto Y. Generation and characterization of motor neuron progenitors and motor neurons using metachromatic leukodystrophy-induced pluripotent stem cells. Mol Genet Metab Rep 2022; 31:100852. [PMID: 35782608 PMCID: PMC9248224 DOI: 10.1016/j.ymgmr.2022.100852] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Accepted: 02/14/2022] [Indexed: 10/29/2022] Open
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Fibbe W, Bernardi R, Charbord P, Krause D, Lo Celso C, Méndez-Ferrer S, Mummery C, Oostendorp R, Raaijmakers M, Socié G, Staal F, Bacigalupo A. The EHA Research Roadmap: Hematopoietic Stem Cells and Allotransplantation. Hemasphere 2022; 6:e0714. [PMID: 35509429 PMCID: PMC9061153 DOI: 10.1097/hs9.0000000000000714] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 03/30/2022] [Indexed: 11/27/2022] Open
Affiliation(s)
- Willem Fibbe
- Department of Internal Medicine and Nephrology, Leiden University Medical Center. Leiden, the Netherlands
| | - Rosa Bernardi
- IRCCS San Raffaele Scientific Institute, Milan, Italy
| | | | - Daniela Krause
- Goethe University Frankfurt and Georg-Speyer-Haus, Frankfurt am Main, Germany
| | - Cristina Lo Celso
- Department of Life Sciences and Centre for Haematology, Imperial College London, United Kingdom
| | | | - Christine Mummery
- Department of Anatomy & Embryology, Leiden University Medical Center, Leiden, the Netherlands
| | - Robert Oostendorp
- Department of Internal Medicine III, Technical University of Munich, School of Medicine, Munich, Germany
| | | | - Gerard Socié
- Hospital Saint Louis, APHP & University of Paris, France
| | - Frank Staal
- Department of Immunology, Leiden University Medical Center, Leiden, the Netherlands
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41
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Rashid SA, Blanchard AT, Combs JD, Fernandez N, Dong Y, Cho HC, Salaita K. DNA Tension Probes Show that Cardiomyocyte Maturation Is Sensitive to the Piconewton Traction Forces Transmitted by Integrins. ACS NANO 2022; 16:5335-5348. [PMID: 35324164 PMCID: PMC11238821 DOI: 10.1021/acsnano.1c04303] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk extracellular matrix stiffness in mediating the functional fate of CMCs. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (∼12, ∼56, and ∼160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. We show that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements, and protein expression (F-actin, vinculin, α-actinin, YAP, and SERCA2a). Additionally, sarcomeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.
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Affiliation(s)
- Sk Aysha Rashid
- Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
| | - Aaron T Blanchard
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, Georgia 30332, United States
| | - J Dale Combs
- Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
| | - Natasha Fernandez
- Division of Pediatric Cardiology, Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, 1405 Clifton Road NE, Atlanta, Georgia 30322, United States
| | - Yixiao Dong
- Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
| | - Hee Cheol Cho
- Division of Pediatric Cardiology, Department of Pediatrics, Emory University School of Medicine and Children's Healthcare of Atlanta, 1405 Clifton Road NE, Atlanta, Georgia 30322, United States
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, Georgia 30332, United States
| | - Khalid Salaita
- Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, Atlanta, Georgia 30332, United States
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42
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Wang J, Chen C, Wang L, Xie M, Ge X, Wu S, He Y, Mou X, Ye C, Sun Y. Patient-Derived Tumor Organoids: New Progress and Opportunities to Facilitate Precision Cancer Immunotherapy. Front Oncol 2022; 12:872531. [PMID: 35449581 PMCID: PMC9016336 DOI: 10.3389/fonc.2022.872531] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Accepted: 03/14/2022] [Indexed: 12/18/2022] Open
Abstract
Cancer immunotherapy has revolutionized the field of cancer treatment in recent years. However, not all patients receiving cancer immunotherapy exhibit durable responses, and reliable, high-throughput testing platforms are urgently needed to guide personalized cancer immunotherapy. The ability of patient-derived tumor organoids to recapitulate pivotal features of original cancer tissues makes them useful as a preclinical model for cancer research and precision medicine. Nevertheless, many challenges exist in the translation of tumor organoid research to clinical decision making. Herein we discuss the applications of patient-derived tumor organoid models and the advances and potential of using complex immune-organoid systems as testing platforms to facilitate precision cancer immunotherapy. In addition, we highlight intriguing applications of tumor organoids with novel multi-omics in preclinical cancer research, highlighting genetic editing, proteomics, and liquid biopsy.
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Affiliation(s)
- Ji Wang
- Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China
| | - Chao Chen
- Department of Colorectal Surgery and Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.,Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.,Cancer Center, Zhejiang University, Hangzhou, China
| | - Lu Wang
- Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China
| | - Mingjun Xie
- State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou, China.,Key Laboratory of Materials Processing and Mold, Zhengzhou University, Zhengzhou, China
| | - Xinyang Ge
- College of Letters and Science, University of California, Los Angeles, Los Angeles, CA, United States
| | - Sufan Wu
- Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China
| | - Yong He
- Cancer Center, Zhejiang University, Hangzhou, China.,State Key Laboratory of Fluid Power and Mechatronic Systems, School of Mechanical Engineering, Zhejiang University, Hangzhou, China.,Key Laboratory of Materials Processing and Mold, Zhengzhou University, Zhengzhou, China.,Key Laboratory of 3D Printing Process and Equipment of Zhejiang Province, College of Mechanical Engineering, Zhejiang University, Hangzhou, China
| | - Xiaozhou Mou
- Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China
| | - Chenyang Ye
- Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China.,Cancer Center, Zhejiang University, Hangzhou, China.,Department of Medical Oncology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China
| | - Yi Sun
- Center for Plastic & Reconstructive Surgery, Department of Plastic & Reconstructive Surgery, Zhejiang Provincial People's Hospital (Affiliated People's Hospital, Hangzhou Medical College), Hangzhou, China
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Vojnits K, Nakanishi M, Porras D, Kim Y, Feng Z, Golubeva D, Bhatia M. Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening. Molecules 2022; 27:molecules27082434. [PMID: 35458632 PMCID: PMC9025795 DOI: 10.3390/molecules27082434] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 04/03/2022] [Accepted: 04/06/2022] [Indexed: 12/22/2022] Open
Abstract
Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.
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Sarkar AK, Nakamura S, Nakai K, Sato T, Shiga T, Abe Y, Hoashi Y, Inoue T, Akamatsu W, Baba K. Increased excitability of human iPSC-derived neurons in HTR2A variant-related sleep bruxism. Stem Cell Res 2022; 59:102658. [PMID: 34999422 DOI: 10.1016/j.scr.2022.102658] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2021] [Revised: 12/05/2021] [Accepted: 01/02/2022] [Indexed: 10/19/2022] Open
Abstract
Sleep bruxism (SB) is a sleep-related movement disorder characterized by grinding and clenching of the teeth during sleep. We previously found a significant association between SB and a single nucleotide polymorphism (SNP), rs6313, in the neuronal serotonin 2A receptor gene (HTR2A), and established human induced pluripotent stem cell (iPSC)-derived neurons from SB patients with a genetic variant. To elucidate the electrophysiological characteristics of SB iPSC-derived neural cells bearing an SB-related genetic variant, we generated ventral hindbrain neurons from SB patients and unaffected controls, and explored the intrinsic membrane properties of these neurons using the patch-clamp technique. We found that the electrophysiological properties of iPSC-derived neurons mature in a time-dependent manner in long-term control cultures. SB neurons exhibited higher action potential firing frequency, higher gain, and shorter action potential half duration. This is the first in vitro modeling of SB using patient-specific iPSCs. The revealed electrophysiological characteristics may serve as a benchmark for further investigation of pathogenic mechanisms underlying SB. Moreover, our results on long-term cultures provide a strategy to define the functional maturity of human neurons in vitro, which can be implemented for stem cell research of neurogenesis, and neurodevelopmental disorders.
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Affiliation(s)
- Avijite Kumer Sarkar
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
| | - Shiro Nakamura
- Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
| | - Kento Nakai
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
| | - Taro Sato
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
| | - Takahiro Shiga
- Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
| | - Yuka Abe
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
| | - Yurie Hoashi
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
| | - Tomio Inoue
- Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
| | - Wado Akamatsu
- Center for Genomic and Regenerative Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
| | - Kazuyoshi Baba
- Department of Prosthodontics, Showa University School of Dentistry, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
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45
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Conversion of Human Fibroblasts into Induced Neural Stem Cells by Small Molecules. Int J Mol Sci 2022; 23:ijms23031740. [PMID: 35163660 PMCID: PMC8835839 DOI: 10.3390/ijms23031740] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2021] [Revised: 02/02/2022] [Accepted: 02/02/2022] [Indexed: 02/05/2023] Open
Abstract
Induced neural stem cells (iNSCs) reprogrammed from somatic cells hold great potentials for drug discovery, disease modelling and the treatment of neurological diseases. Although studies have shown that human somatic cells can be converted into iNSCs by introducing transcription factors, these iNSCs are unlikely to be used for clinical application due to the safety concern of using exogenous genes and viral transduction vectors. Here, we report the successful conversion of human fibroblasts into iNSCs using a cocktail of small molecules. Furthermore, our results demonstrate that these human iNSCs (hiNSCs) have similar gene expression profiles to bona fide NSCs, can proliferate, and are capable of differentiating into glial cells and functional neurons. This study collectively describes a novel approach based on small molecules to produce hiNSCs from human fibroblasts, which may be useful for both research and therapeutic purposes.
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46
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Ajalik RE, Alenchery RG, Cognetti JS, Zhang VZ, McGrath JL, Miller BL, Awad HA. Human Organ-on-a-Chip Microphysiological Systems to Model Musculoskeletal Pathologies and Accelerate Therapeutic Discovery. Front Bioeng Biotechnol 2022; 10:846230. [PMID: 35360391 PMCID: PMC8964284 DOI: 10.3389/fbioe.2022.846230] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2021] [Accepted: 02/21/2022] [Indexed: 12/12/2022] Open
Abstract
Human Microphysiological Systems (hMPS), otherwise known as organ- and tissue-on-a-chip models, are an emerging technology with the potential to replace in vivo animal studies with in vitro models that emulate human physiology at basic levels. hMPS platforms are designed to overcome limitations of two-dimensional (2D) cell culture systems by mimicking 3D tissue organization and microenvironmental cues that are physiologically and clinically relevant. Unlike animal studies, hMPS models can be configured for high content or high throughput screening in preclinical drug development. Applications in modeling acute and chronic injuries in the musculoskeletal system are slowly developing. However, the complexity and load bearing nature of musculoskeletal tissues and joints present unique challenges related to our limited understanding of disease mechanisms and the lack of consensus biomarkers to guide biological therapy development. With emphasis on examples of modeling musculoskeletal tissues, joints on chips, and organoids, this review highlights current trends of microphysiological systems technology. The review surveys state-of-the-art design and fabrication considerations inspired by lessons from bioreactors and biological variables emphasizing the role of induced pluripotent stem cells and genetic engineering in creating isogenic, patient-specific multicellular hMPS. The major challenges in modeling musculoskeletal tissues using hMPS chips are identified, including incorporating biological barriers, simulating joint compartments and heterogenous tissue interfaces, simulating immune interactions and inflammatory factors, simulating effects of in vivo loading, recording nociceptors responses as surrogates for pain outcomes, modeling the dynamic injury and healing responses by monitoring secreted proteins in real time, and creating arrayed formats for robotic high throughput screens. Overcoming these barriers will revolutionize musculoskeletal research by enabling physiologically relevant, predictive models of human tissues and joint diseases to accelerate and de-risk therapeutic discovery and translation to the clinic.
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Affiliation(s)
- Raquel E. Ajalik
- Center for Musculoskeletal Research, University of Rochester, Rochester, NY, United States
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Rahul G. Alenchery
- Center for Musculoskeletal Research, University of Rochester, Rochester, NY, United States
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - John S. Cognetti
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Victor Z. Zhang
- Center for Musculoskeletal Research, University of Rochester, Rochester, NY, United States
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - James L. McGrath
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
| | - Benjamin L. Miller
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
- Department of Dermatology, University of Rochester, Rochester, NY, United States
| | - Hani A. Awad
- Center for Musculoskeletal Research, University of Rochester, Rochester, NY, United States
- Department of Biomedical Engineering, University of Rochester, Rochester, NY, United States
- *Correspondence: Hani A. Awad,
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Smith VM, Nguyen H, Rumsey JW, Long CJ, Shuler ML, Hickman JJ. A Functional Human-on-a-Chip Autoimmune Disease Model of Myasthenia Gravis for Development of Therapeutics. Front Cell Dev Biol 2021; 9:745897. [PMID: 34881241 PMCID: PMC8645836 DOI: 10.3389/fcell.2021.745897] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Accepted: 10/14/2021] [Indexed: 11/13/2022] Open
Abstract
Myasthenia gravis (MG) is a chronic and progressive neuromuscular disease where autoantibodies target essential proteins such as the nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction (NMJ) causing muscle fatigue and weakness. Autoantibodies directed against nAChRs are proposed to work by three main pathological mechanisms of receptor disruption: blocking, receptor internalization, and downregulation. Current in vivo models using experimental autoimmune animal models fail to recapitulate the disease pathology and are limited in clinical translatability due to disproportionate disease severity and high animal death rates. The development of a highly sensitive antibody assay that mimics human disease pathology is desirable for clinical advancement and therapeutic development. To address this lack of relevant models, an NMJ platform derived from human iPSC differentiated motoneurons and primary skeletal muscle was used to investigate the ability of an anti-nAChR antibody to induce clinically relevant MG pathology in the serum-free, spatially organized, functionally mature NMJ platform. Treatment of the NMJ model with the anti-nAChR antibody revealed decreasing NMJ stability as measured by the number of NMJs before and after the synchrony stimulation protocol. This decrease in NMJ stability was dose-dependent over a concentration range of 0.01-20 μg/mL. Immunocytochemical (ICC) analysis was used to distinguish between pathological mechanisms of antibody-mediated receptor disruption including blocking, receptor internalization and downregulation. Antibody treatment also activated the complement cascade as indicated by complement protein 3 deposition near the nAChRs. Additionally, complement cascade activation significantly altered other readouts of NMJ function including the NMJ fidelity parameter as measured by the number of muscle contractions missed in response to increasing motoneuron stimulation frequencies. This synchrony readout mimics the clinical phenotype of neurological blocking that results in failure of muscle contractions despite motoneuron stimulations. Taken together, these data indicate the establishment of a relevant disease model of MG that mimics reduction of functional nAChRs at the NMJ, decreased NMJ stability, complement activation and blocking of neuromuscular transmission. This system is the first functional human in vitro model of MG to be used to simulate three potential disease mechanisms as well as to establish a preclinical platform for evaluation of disease modifying treatments (etiology).
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Affiliation(s)
- Virginia M. Smith
- Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, FL, United States
- Hesperos, Inc., Orlando, FL, United States
| | - Huan Nguyen
- Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, FL, United States
| | | | | | | | - James J. Hickman
- Hybrid Systems Lab, NanoScience Technology Center, University of Central Florida, Orlando, FL, United States
- Hesperos, Inc., Orlando, FL, United States
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48
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Disruption of Mitochondrial Homeostasis: The Role of PINK1 in Parkinson's Disease. Cells 2021; 10:cells10113022. [PMID: 34831247 PMCID: PMC8616241 DOI: 10.3390/cells10113022] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2021] [Revised: 10/29/2021] [Accepted: 11/01/2021] [Indexed: 12/18/2022] Open
Abstract
The progressive reduction of the dopaminergic neurons of the substantia nigra is the fundamental process underlying Parkinson’s disease (PD), while the mechanism of susceptibility of this specific neuronal population is largely unclear. Disturbances in mitochondrial function have been recognized as one of the main pathways in sporadic PD since the finding of respiratory chain impairment in animal models of PD. Studies on genetic forms of PD have provided new insight on the role of mitochondrial bioenergetics, homeostasis, and autophagy. PINK1 (PTEN-induced putative kinase 1) gene mutations, although rare, are the second most common cause of recessively inherited early-onset PD, after Parkin gene mutations. Our knowledge of PINK1 and Parkin function has increased dramatically in the last years, with the discovery that a process called mitophagy, which plays a key role in the maintenance of mitochondrial health, is mediated by the PINK1/Parkin pathway. In vitro and in vivo models have been developed, supporting the role of PINK1 in synaptic transmission, particularly affecting dopaminergic neurons. It is of paramount importance to further define the role of PINK1 in mitophagy and mitochondrial homeostasis in PD pathogenesis in order to delineate novel therapeutic targets.
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49
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Yi SA, Zhang Y, Rathnam C, Pongkulapa T, Lee KB. Bioengineering Approaches for the Advanced Organoid Research. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2021; 33:e2007949. [PMID: 34561899 PMCID: PMC8682947 DOI: 10.1002/adma.202007949] [Citation(s) in RCA: 72] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 06/09/2021] [Indexed: 05/09/2023]
Abstract
Recent advances in 3D cell culture technology have enabled scientists to generate stem cell derived organoids that recapitulate the structural and functional characteristics of native organs. Current organoid technologies have been striding toward identifying the essential factors for controlling the processes involved in organoid development, including physical cues and biochemical signaling. There is a growing demand for engineering dynamic niches characterized by conditions that resemble in vivo organogenesis to generate reproducible and reliable organoids for various applications. Innovative biomaterial-based and advanced engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of organoid research. The recent advances in organoid engineering, including extracellular matrices and genetic modulation, are comprehensively summarized to pinpoint the parameters critical for organ-specific patterning. Moreover, perspective trends in developing tunable organoids in response to exogenous and endogenous cues are discussed for next-generation developmental studies, disease modeling, and therapeutics.
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Affiliation(s)
- Sang Ah Yi
- Epigenome Dynamics Control Research Center, School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, 16419, Republic of Korea
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ, 08854, USA
| | - Yixiao Zhang
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ, 08854, USA
| | - Christopher Rathnam
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ, 08854, USA
| | - Thanapat Pongkulapa
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ, 08854, USA
| | - Ki-Bum Lee
- Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 123 Bevier Road, Piscataway, NJ, 08854, USA
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50
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Optimization of Neurite Tracing and Further Characterization of Human Monocyte-Derived-Neuronal-like Cells. Brain Sci 2021; 11:brainsci11111372. [PMID: 34827371 PMCID: PMC8615477 DOI: 10.3390/brainsci11111372] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2021] [Revised: 10/14/2021] [Accepted: 10/14/2021] [Indexed: 12/17/2022] Open
Abstract
Deficits in neuronal structure are consistently associated with neurodevelopmental illnesses such as autism and schizophrenia. Nonetheless, the inability to access neurons from clinical patients has limited the study of early neurostructural changes directly in patients’ cells. This obstacle has been circumvented by differentiating stem cells into neurons, although the most used methodologies are time consuming. Therefore, we recently developed a relatively rapid (~20 days) protocol for transdifferentiating human circulating monocytes into neuronal-like cells. These monocyte-derived-neuronal-like cells (MDNCs) express several genes and proteins considered neuronal markers, such as MAP-2 and PSD-95. In addition, these cells conduct electrical activity. We have also previously shown that the structure of MDNCs is comparable with that of human developing neurons (HDNs) after 5 days in culture. Moreover, the neurostructure of MDNCs responds similarly to that of HDNs when exposed to colchicine and dopamine. In this manuscript, we expanded our characterization of MDNCs to include the expression of 12 neuronal genes, including tau. Following, we compared three different tracing approaches (two semi-automated and one automated) that enable tracing using photographs of live cells. This comparison is imperative for determining which neurite tracing method is more efficient in extracting neurostructural data from MDNCs and thus allowing researchers to take advantage of the faster yield provided by these neuronal-like cells. Surprisingly, it was one of the semi-automated methods that was the fastest, consisting of tracing only the longest primary and the longest secondary neurite. This tracing technique also detected more structural deficits. The only automated method tested, Volocity, detected MDNCs but failed to trace the entire neuritic length. Other advantages and disadvantages of the three tracing approaches are also presented and discussed.
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