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Lica JJ, Jakóbkiewicz-Banecka J, Hellmann A. In Vitro models of leukemia development: the role of very small leukemic stem-like cells in the cellular transformation cascade. Front Cell Dev Biol 2025; 12:1463807. [PMID: 39830209 PMCID: PMC11740207 DOI: 10.3389/fcell.2024.1463807] [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: 07/15/2024] [Accepted: 11/28/2024] [Indexed: 01/22/2025] Open
Abstract
Recent experimental findings indicate that cancer stem cells originate from transformed very small embryonic-like stem cells. This finding represents an essential advancement in uncovering the processes that drive the onset and progression of cancer. In continuously growing cell lines, for the first time, our team's follow-up research on leukemia, lung cancer, and healthy embryonic kidney cells revealed stages that resembles very small precursor stem cells. This review explores the origin of leukemic stem-like cells from very small leukemic stem-like cells establish from transformed very small embryonic-like stem cells. We explore theoretical model of acute myeloid leukemia initiation and progresses through various stages, as well basing the HL60 cell line, present its hierarchical stage development in vitro, highlighting the role of these very small precursor primitive stages. We also discuss the potential implications of further research into these unique cellular stages for advancing leukemia and cancer treatment and prevention.
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Affiliation(s)
- Jan Jakub Lica
- Department Medical Biology and Genetics, Faculty of Biology, University of Gdansk, Gdansk, Poland
- Department Health Science; Powiśle University, Gdańsk, Poland
| | | | - Andrzej Hellmann
- Department of Hematology and Transplantology, Faculty of Medicine, Medical University of Gdansk, Gdańsk, Poland
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Pierro M, Thébaud B. Cell-based strategies for the treatment of injury to the developing lung. THE LUNG 2025:403-426. [DOI: 10.1016/b978-0-323-91824-4.00020-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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de La Bourdonnaye G, Marek M, Ghazalova T, Damborsky J, Pachl P, Brynda J, Stepankova V, Chaloupkova R. Structural analysis of the stable form of fibroblast growth factor 2 - FGF2-STAB. J Struct Biol X 2024; 10:100112. [PMID: 39512606 PMCID: PMC11541812 DOI: 10.1016/j.yjsbx.2024.100112] [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: 07/04/2024] [Revised: 09/24/2024] [Accepted: 10/14/2024] [Indexed: 11/15/2024] Open
Abstract
Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.
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Affiliation(s)
- Gabin de La Bourdonnaye
- Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic
- Enantis Ltd., Biotechnology Incubator INBIT, Brno, Czech Republic
| | - Martin Marek
- Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic
- International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic
| | - Tereza Ghazalova
- Enantis Ltd., Biotechnology Incubator INBIT, Brno, Czech Republic
| | - Jiri Damborsky
- Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic
- International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic
| | - Petr Pachl
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Jiri Brynda
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | | | - Radka Chaloupkova
- Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic
- Enantis Ltd., Biotechnology Incubator INBIT, Brno, Czech Republic
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Chandrababu A, Puthumana J. CRISPR-edited, cell-based future-proof meat and seafood to enhance global food security and nutrition. Cytotechnology 2024; 76:619-652. [PMID: 39435422 PMCID: PMC11490478 DOI: 10.1007/s10616-024-00645-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Accepted: 07/15/2024] [Indexed: 10/23/2024] Open
Abstract
Food security is a major concern due to the growing population and climate change. A method for increasing food production is the use of modern biotechnology, such as cell culture, marker-assisted selection, and genetic engineering. Cellular agriculture has enabled the production of cell-cultivated meat in bioreactors that mimic the properties of conventional meat. Furthermore, 3D food printing technology has improved food production by adding new nutritional and organoleptic properties. Marker-assisted selection and genetic engineering could play an important role in producing animals and crops with desirable traits. Therefore, integrating cellular agriculture with genetic engineering technology could be a potential strategy for the production of cell-based meat and seafood with high health benefits in the future. This review highlights the production of cell-cultivated meat derived from a variety of species, including livestock, birds, fish, and marine crustaceans. It also investigates the application of genetic engineering methods, such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein), in the context of cellular agriculture. Moreover, it examines aspects such as food safety, regulatory considerations, and consumer acceptance of genetically engineered cell-cultivated meat and seafood.
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Affiliation(s)
- Aswathy Chandrababu
- National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Cochin, Kerala 16 India
| | - Jayesh Puthumana
- National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Cochin, Kerala 16 India
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Han J, Chear S, Talbot J, Swier V, Booth C, Reuben-Thomas C, Dalvi S, Weimer JM, Hewitt AW, Cook AL, Singh R. Genetic and Cellular Basis of Impaired Phagocytosis and Photoreceptor Degeneration in CLN3 Disease. Invest Ophthalmol Vis Sci 2024; 65:23. [PMID: 39535788 PMCID: PMC11563035 DOI: 10.1167/iovs.65.13.23] [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: 06/13/2024] [Accepted: 10/09/2024] [Indexed: 11/16/2024] Open
Abstract
Purpose CLN3 Batten disease (also known as juvenile neuronal ceroid lipofuscinosis) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease induced pluripotent stem cell-RPE cells show defective phagocytosis of photoreceptor outer segment (POS). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Methods Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3Δ7-8/Δ7-8 (CLN3) Yucatan miniswine was also used to study the impact of CLN3Δ7-8/Δ7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3Δ7-8/Δ7-8 and wild-type miniswine eyes were carried out at 6, 36, or 48 months of age. Results CLN3Δ7-8/Δ7-8 RPE (CLN3 RPE) displayed decreased POS binding and consequently decreased uptake of POS compared with isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3Δ7-8/Δ7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months of age and was followed by almost complete loss of photoreceptors at 48 months of age. Conclusions CLN3Δ7-8/Δ7-8 mutation (which affects ≤85% of patients) affects both RPE and POS and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.
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Affiliation(s)
- Jimin Han
- Department of Ophthalmology, University of Rochester, Rochester, New York, United States
- Department of Biomedical Genetics, University of Rochester, Rochester, New York, United States
- Center for Visual Science, University of Rochester, Rochester, New York, United States
| | - Sueanne Chear
- Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia
| | - Jana Talbot
- Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia
| | - Vicki Swier
- Pediatrics & Rare Diseases Group; Sanford Research, Sioux Falls, South Dakota, United States
| | - Clarissa Booth
- Pediatrics & Rare Diseases Group; Sanford Research, Sioux Falls, South Dakota, United States
| | - Cheyenne Reuben-Thomas
- Department of Ophthalmology, University of Rochester, Rochester, New York, United States
- Department of Biomedical Genetics, University of Rochester, Rochester, New York, United States
- Center for Visual Science, University of Rochester, Rochester, New York, United States
| | - Sonal Dalvi
- Department of Ophthalmology, University of Rochester, Rochester, New York, United States
- Department of Biomedical Genetics, University of Rochester, Rochester, New York, United States
- Center for Visual Science, University of Rochester, Rochester, New York, United States
| | - Jill M. Weimer
- Pediatrics & Rare Diseases Group; Sanford Research, Sioux Falls, South Dakota, United States
- Department of Pediatrics; Sanford School of Medicine, University of South Dakota, Sioux Falls, South Dakota, United States
| | - Alex W. Hewitt
- Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia
| | - Anthony L. Cook
- Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia
| | - Ruchira Singh
- Department of Ophthalmology, University of Rochester, Rochester, New York, United States
- Department of Biomedical Genetics, University of Rochester, Rochester, New York, United States
- Center for Visual Science, University of Rochester, Rochester, New York, United States
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Chen S, Hayoun-Neeman D, Nagar M, Pinyan S, Hadad L, Yaacobov L, Alon L, Shachar LE, Swissa T, Kryukov O, Gershoni-Yahalom O, Rosental B, Cohen S, Lichtenstein RG. Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions. EMBO Rep 2024; 25:4433-4464. [PMID: 39256596 PMCID: PMC11467398 DOI: 10.1038/s44319-024-00243-1] [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: 11/13/2023] [Revised: 08/09/2024] [Accepted: 08/23/2024] [Indexed: 09/12/2024] Open
Abstract
The embryonic cell surface is rich in glycosphingolipids (GSLs), which change during differentiation. The reasons for GSL subgroup variation during early embryogenesis remain elusive. By combining genomic approaches, flow cytometry, confocal imaging, and transcriptomic data analysis, we discovered that α1,2-fucosylated GSLs control the differentiation of human pluripotent cells (hPCs) into germ layer tissues. Overexpression of α1,2-fucosylated GSLs disrupts hPC differentiation into mesodermal lineage and reduces differentiation into cardiomyocytes. Conversely, reducing α1,2-fucosylated groups promotes hPC differentiation and mesoderm commitment in response to external signals. We find that bone morphogenetic protein 4 (BMP4), a mesodermal gene inducer, suppresses α1,2-fucosylated GSL expression. Overexpression of α1,2-fucosylated GSLs impairs SMAD activation despite BMP4 presence, suggesting α-fucosyl end groups as BMP pathway regulators. Additionally, the absence of α1,2-fucosylated GSLs in early/late mesoderm and primitive streak stages in mouse embryos aligns with the hPC results. Thus, α1,2-fucosylated GSLs may regulate early cell-fate decisions and embryo development by modulating cell signaling.
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Affiliation(s)
- Saray Chen
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Dana Hayoun-Neeman
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Michal Nagar
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Sapir Pinyan
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Limor Hadad
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Liat Yaacobov
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Lilach Alon
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Liraz Efrat Shachar
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Tair Swissa
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Olga Kryukov
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Orly Gershoni-Yahalom
- Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Benyamin Rosental
- Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
- The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Smadar Cohen
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
- Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
- The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel
| | - Rachel G Lichtenstein
- Avram and Stella Goren-Goldstein Department of Biotechnology Engineering, Faculty of Engineering Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel.
- Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev, Beer-Sheva, 8410501, Israel.
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Mousavi Mirkalaei S, Farivar S. Systematic optimization of culture media for maintenance of human induced pluripotent stem cells using the response surface methodology. Heliyon 2024; 10:e32558. [PMID: 38975108 PMCID: PMC11226774 DOI: 10.1016/j.heliyon.2024.e32558] [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: 09/16/2023] [Revised: 06/05/2024] [Accepted: 06/05/2024] [Indexed: 07/09/2024] Open
Abstract
The application of human induced pluripotent stem cells (hiPSCs) provides tremendous opportunities in cell therapy. However, culturing these cells faces many practical challenges, including costs associated with cell culture media and the optimization of cell culture conditions. Providing an optimized culture platform for hiPSCs to maintain pluripotency and self-renewal and generate cost-effective and robust therapeutics is an immediate requirement. This study used the design of experiments and the response surface methodology, a powerful statistical tool, to generate empirical models for predicting the optimal culture conditions of the hiPSCs. Pluripotency and cell proliferation were applied as read-outs to determine the optimal concentration of basic fibroblast growth factor (bFGF) and cell density. One model was defined to predict pluripotency and cell proliferation in terms of the predictor variables of the bFGF concentration and cell seeding density. Predicted culture conditions to maximize maintaining cell pluripotency were successfully validated. The present study's findings provide a novel approach that can potentially allow controllable hiPSC culture routine in translational research.
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Affiliation(s)
- Seyedmilad Mousavi Mirkalaei
- Department of Cell and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
| | - Shirin Farivar
- Department of Cell and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
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Zhao S, Li J, Duan S, Liu C, Wang H, Lu J, Zhao N, Sheng X, Wu Y, Li Y, Sun B, Liu L. UBQLN1 links proteostasis and mitochondria function to telomere maintenance in human embryonic stem cells. Stem Cell Res Ther 2024; 15:180. [PMID: 38902824 PMCID: PMC11191273 DOI: 10.1186/s13287-024-03789-y] [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: 01/18/2024] [Accepted: 06/09/2024] [Indexed: 06/22/2024] Open
Abstract
BACKGROUND Telomeres consist of repetitive DNA sequences at the chromosome ends to protect chromosomal stability, and primarily maintained by telomerase or occasionally by alternative telomere lengthening of telomeres (ALT) through recombination-based mechanisms. Additional mechanisms that may regulate telomere maintenance remain to be explored. Simultaneous measurement of telomere length and transcriptome in the same human embryonic stem cell (hESC) revealed that mRNA expression levels of UBQLN1 exhibit linear relationship with telomere length. METHODS In this study, we first generated UBQLN1-deficient hESCs and compared with the wild-type (WT) hESCs the telomere length and molecular change at RNA and protein level by RNA-seq and proteomics. Then we identified the potential interacting proteins with UBQLN1 using immunoprecipitation-mass spectrometry (IP-MS). Furthermore, the potential mechanisms underlying the shortened telomeres in UBQLN1-deficient hESCs were analyzed. RESULTS We show that Ubiquilin1 (UBQLN1) is critical for telomere maintenance in human embryonic stem cells (hESCs) via promoting mitochondrial function. UBQLN1 deficiency leads to oxidative stress, loss of proteostasis, mitochondria dysfunction, DNA damage, and telomere attrition. Reducing oxidative damage and promoting mitochondria function by culture under hypoxia condition or supplementation with N-acetylcysteine partly attenuate the telomere attrition induced by UBQLN1 deficiency. Moreover, UBQLN1 deficiency/telomere shortening downregulates genes for neuro-ectoderm lineage differentiation. CONCLUSIONS Altogether, UBQLN1 functions to scavenge ubiquitinated proteins, preventing their overloading mitochondria and elevated mitophagy. UBQLN1 maintains mitochondria and telomeres by regulating proteostasis and plays critical role in neuro-ectoderm differentiation.
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Affiliation(s)
- Shuang Zhao
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Jie Li
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Songqi Duan
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Chang Liu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Hua Wang
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Jiangtao Lu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Nannan Zhao
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Xiaoyan Sheng
- Experimental Animal Center, Nankai University, Tianjin, 300350, China
| | - Yiwei Wu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Yanjun Li
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Baofa Sun
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China
| | - Lin Liu
- State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300350, China.
- Frontiers Science Center for Cell Responses, College of Life Science, Nankai University, Tianjin, 300071, China.
- Experimental Animal Center, Nankai University, Tianjin, 300350, China.
- Tianjin Union Medical Center, Nankai University, Tianjin, 300071, China.
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Han J, Chear S, Talbot J, Swier V, Booth C, Reuben-Thomas C, Dalvi S, Weimer JM, Hewitt AW, Cook AL, Singh R. Genetic and cellular basis of impaired phagocytosis and photoreceptor degeneration in CLN3 disease. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.09.597388. [PMID: 38895469 PMCID: PMC11185776 DOI: 10.1101/2024.06.09.597388] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/21/2024]
Abstract
Purpose CLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between CLN3 mutation and POS phagocytosis defect. Methods Isogenic control and CLN3 mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic CLN3 Δ 7-8/ Δ 7-8 ( CLN3 ) Yucatan miniswine was also used to study the impact of CLN3 Δ 7-8/ Δ 7-8 mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of CLN3 Δ 7/8 and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age. Results CLN3 Δ 7-8/ Δ 7-8 RPE ( CLN3 RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed CLN3 Δ 7-8/ Δ 7-8 POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in CLN3 miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age. Conclusions CLN3 Δ 7-8/ Δ 7-8 mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.
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Yanagihara K, Hayashi Y, Liu Y, Yamaguchi T, Hemmi Y, Kokunugi M, Yamada KU, Fukumoto K, Suga M, Terada S, Nikawa H, Kawabata K, Furue M. Trisomy 12 compromises the mesendodermal differentiation propensity of human pluripotent stem cells. In Vitro Cell Dev Biol Anim 2024; 60:521-534. [PMID: 38169039 PMCID: PMC11126453 DOI: 10.1007/s11626-023-00824-9] [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: 08/12/2023] [Accepted: 09/08/2023] [Indexed: 01/05/2024]
Abstract
Trisomy 12 is one of the most frequent chromosomal abnormalities in cultured human pluripotent stem cells (hPSCs). Although potential oncogenic properties and augmented cell cycle caused by trisomy 12 have been reported, the consequences of trisomy 12 in terms of cell differentiation, which is the basis for regenerative medicine, drug development, and developmental biology studies, have not yet been investigated. Here, we report that trisomy 12 compromises the mesendodermal differentiation of hPSCs. We identified sublines of hPSCs carrying trisomy 12 after their prolonged culture. Transcriptome analysis revealed that these hPSC sublines carried abnormal gene expression patterns in specific signaling pathways in addition to cancer-related cell cycle pathways. These hPSC sublines showed a lower propensity for mesendodermal differentiation in embryoid bodies cultured in a serum-free medium. BMP4-induced exit from the self-renewal state was impaired in the trisomy 12 hPSC sublines, with less upregulation of key transcription factor gene expression. As a consequence, the differentiation efficiency of hematopoietic and hepatic lineages was also impaired in the trisomy 12 hPSC sublines. We reveal that trisomy 12 disrupts the genome-wide expression patterns that are required for proper mesendodermal differentiation.
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Affiliation(s)
- Kana Yanagihara
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yohei Hayashi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
| | - Yujung Liu
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Tomoko Yamaguchi
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Yasuko Hemmi
- iPS Cell Advanced Characterization and Development Team, RIKEN Bioresource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan
| | - Minako Kokunugi
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kozue Uchio Yamada
- Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Ken Fukumoto
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Mika Suga
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Satoshi Terada
- Department of Applied Chemistry and Biotechnology, University of Fukui, Fukui City, 3-9-1 Bunkyo, Fukui, 910-8507, Japan
| | - Hiroki Nikawa
- Department of Oral Biology & Engineering Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Kenji Kawabata
- Laboratory of Cell Model for Drug Discovery, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan
| | - Miho Furue
- Laboratory of Stem Cell Cultures, National Institutes of Biomedical Innovation, Health, and Nutrition, 7-6-8, Saito-Asagi, Osaka, Ibaraki, 567-0085, Japan.
- Cel-MiM, Ltd., Tokyo, Japan.
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11
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Hagemann C, Bailey MCD, Carraro E, Stankevich KS, Lionello VM, Khokhar N, Suklai P, Moreno-Gonzalez C, O’Toole K, Konstantinou G, Dix CL, Joshi S, Giagnorio E, Bergholt MS, Spicer CD, Imbert A, Tedesco FS, Serio A. Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs. PLoS Biol 2024; 22:e3002503. [PMID: 38478490 PMCID: PMC10936828 DOI: 10.1371/journal.pbio.3002503] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2023] [Accepted: 01/17/2024] [Indexed: 03/17/2024] Open
Abstract
Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.
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Affiliation(s)
- Cathleen Hagemann
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
| | - Matthew C. D. Bailey
- The Francis Crick Institute, London, United Kingdom
- Centre for Craniofacial & Regenerative Biology, King’s College London, London, United Kingdom
| | - Eugenia Carraro
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
| | - Ksenia S. Stankevich
- Department of Chemistry and York Biomedical Research Institute, University of York, York, United Kingdom
| | - Valentina Maria Lionello
- The Francis Crick Institute, London, United Kingdom
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
| | - Noreen Khokhar
- The Francis Crick Institute, London, United Kingdom
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
- Randall Centre for Cell and Molecular Biophysics, King’s College London, London, United Kingdom
| | - Pacharaporn Suklai
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
| | - Carmen Moreno-Gonzalez
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
| | - Kelly O’Toole
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
| | | | | | - Sudeep Joshi
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
| | - Eleonora Giagnorio
- The Francis Crick Institute, London, United Kingdom
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
- Neurology IV—Neuroimmunology and Neuromuscular Diseases Unit, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, Italy
| | - Mads S. Bergholt
- Centre for Craniofacial & Regenerative Biology, King’s College London, London, United Kingdom
| | - Christopher D. Spicer
- Department of Chemistry and York Biomedical Research Institute, University of York, York, United Kingdom
| | | | - Francesco Saverio Tedesco
- The Francis Crick Institute, London, United Kingdom
- Department of Cell and Developmental Biology, University College London, London, United Kingdom
- Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital for Children, London, United Kingdom
| | - Andrea Serio
- United Kingdom Dementia Research Institute Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, Maurice Wohl Clinical Neuroscience Institute, London, United Kingdom
- The Francis Crick Institute, London, United Kingdom
- Dementia Research Institute (UK DRI)
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12
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Kajihara R, Ezaki R, Ichikawa K, Watanabe T, Terada T, Matsuzaki M, Horiuchi H. Wnt signaling blockade is essential for maintaining the pluripotency of chicken embryonic stem cells. Poult Sci 2024; 103:103361. [PMID: 38154448 PMCID: PMC10788285 DOI: 10.1016/j.psj.2023.103361] [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: 10/07/2023] [Revised: 12/04/2023] [Accepted: 12/04/2023] [Indexed: 12/30/2023] Open
Abstract
Activation of Wnt/β-catenin signaling supports the self-renewal of mouse embryonic stem cells. We aimed to understand the effects of Wnt signaling activation or inhibition on chicken embryonic stem cells (chESCs), as these effects are largely unknown. When the glycogen synthase kinase-3 β inhibitor CHIR99021-which activates Wnt signaling-was added to chESC cultures, the colony shape flattened, and the expression levels of pluripotency-related (NANOG, SOX2, SOX3, OCT4, LIN28A, DNMT3B, and PRDM14) and germ cell (CVH and DAZL) markers showed a decreasing trend, and the growth of chESCs was inhibited after approximately 7 d. By contrast, when the Wnt signaling inhibitor XAV939 was added to the culture, dense and compact multipotent colonies (morphologically similar to mouse embryonic stem cell colonies) showing stable expression of pluripotency-related and germline markers were formed. The addition of XAV939 stabilized the proliferation of chESCs in the early stages of culture and promoted their establishment. Furthermore, these chESCs formed chimeras. In conclusion, functional chESCs can be stably cultured using Wnt signaling inhibitors. These findings suggest the importance of Wnt/β-catenin signaling in avian stem cells, offering valuable insights for applied research using chESCs.
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Affiliation(s)
- Ryota Kajihara
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
| | - Ryo Ezaki
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
| | - Kennosuke Ichikawa
- The Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, United Kingdom
| | - Tenkai Watanabe
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
| | - Takumi Terada
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
| | - Mei Matsuzaki
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
| | - Hiroyuki Horiuchi
- Laboratory of Immunobiology, Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; Genome Editing Innovation Center, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
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13
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Ahn Y, Jeong J, Choi KH, Lee DK, Lee M, Lee NY, Kim DY, Lee CK. Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer. Stem Cells Dev 2023; 32:747-757. [PMID: 37756363 DOI: 10.1089/scd.2023.0171] [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] [Indexed: 09/29/2023] Open
Abstract
Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.
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Affiliation(s)
- Yelim Ahn
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
| | - Jinsol Jeong
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
| | - Kwang-Hwan Choi
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
- Research and Development Center, Space F Corporation, Hwasung, South Korea
| | - Dong-Kyung Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
- Research and Development Center, Space F Corporation, Hwasung, South Korea
| | - Mingyun Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
| | - Na-Young Lee
- Department of Veterinary Pathology, College of Veterinary Medicine; Seoul National University, Seoul, South Korea
| | - Dae-Yong Kim
- Department of Veterinary Pathology, College of Veterinary Medicine; Seoul National University, Seoul, South Korea
| | - Chang-Kyu Lee
- Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science; College of Veterinary Medicine; Seoul National University, Seoul, South Korea
- Institute of Green Bio Science and Technology, Seoul National University, Pyeongchang, South Korea
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14
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Choi EW, Lim IR, Park JH, Song J, Choi B, Kim S. Exosomes derived from mesenchymal stem cells primed with disease-condition-serum improved therapeutic efficacy in a mouse rheumatoid arthritis model via enhanced TGF-β1 production. Stem Cell Res Ther 2023; 14:283. [PMID: 37794417 PMCID: PMC10552321 DOI: 10.1186/s13287-023-03523-0] [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: 07/19/2023] [Accepted: 09/26/2023] [Indexed: 10/06/2023] Open
Abstract
BACKGROUNDS Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation-mediated progressive destruction of the cartilage and bone, resulting in reduced quality of life. We primed human telomerase reverse transcriptase-overexpressing immortalized human adipose tissue-derived mesenchymal stem cells (iMSCs) with serum derived from a non-human primate RA model and studied the immunomodulatory ability of exosomes obtained from primed iMSCs. METHODS After immunophenotyping, nanoparticle tracking analysis, and in vitro functional tests, Dulbecco's phosphate-buffered saline (dPBS, Group C), exosomes derived from the supernatant of iMSCs (Exo-FBS, Group E), exosomes derived from the supernatant of iMSCs primed with RA serum (Exo-RA, Group F), and methotrexate (Group M) were administered in collagen-induced arthritis (CIA) model mice. dPBS was administered to the normal (N) group for comparison (n = 10/group). RESULTS Exo-RA had a significantly higher number of exosomes compared to Exo-FBS when measured with nanoparticle tracking analysis or exosome marker CD81, and Transforming growth factor-β1 amounts were significantly higher in Exo-RA than in Exo-FBS. When Exo-FBS or Exo-RA was administered to the collagen-induced arthritis model, serum interleukin (IL)-4 and the proportion of Th2 (CD4+CD25+GATA3+) and M2 (CD11c - CD206+ of CD45+CD64+) cells were significantly increased compared to the control group. Furthermore, Exo-RA could alleviate cartilage damage by significantly lowering the concentrations of proinflammatory cytokines such as tumor necrosis factor-α, keratinocyte chemoattractant, and IL-12p70. CONCLUSION Exosomes derived from disease-condition-serum-primed iMSCs ameliorated cartilage damage in a RA model by enhancing TGF-β1 production, inducing Th2 and M2 polarization and lowering proinflammatory cytokines, TNF-α, KC, and IL-12p70 in the host. Patient-derived serum can be used as an iMSC priming strategy in iMSC-derived exosome treatment of RA.
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Affiliation(s)
- Eun Wha Choi
- Department of Veterinary Clinical Pathology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon, Gangwon-do, 24341, Republic of Korea.
| | - I-Rang Lim
- Department of Veterinary Clinical Pathology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon, Gangwon-do, 24341, Republic of Korea
| | - Ji Hong Park
- Department of Veterinary Clinical Pathology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, 1 Kangwondaehak-gil, Chuncheon, Gangwon-do, 24341, Republic of Korea
| | - Jiwoo Song
- Bioanalysis Center, GenNBio Inc., 700, Daewangpangyo-ro, Bundang-guGyeonggi-do, Seongnam-si, 13488, Republic of Korea
| | - Bongkum Choi
- Bioanalysis Center, GenNBio Inc., 700, Daewangpangyo-ro, Bundang-guGyeonggi-do, Seongnam-si, 13488, Republic of Korea
| | - Sungjoo Kim
- GenNBio Inc., 80, Deurimsandan 2-ro, Cheongbuk-eup, Pyeongtaek-si, Gyeonggi-do, 17796, Republic of Korea
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15
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Kulus M, Jankowski M, Kranc W, Golkar Narenji A, Farzaneh M, Dzięgiel P, Zabel M, Antosik P, Bukowska D, Mozdziak P, Kempisty B. Bioreactors, scaffolds and microcarriers and in vitro meat production-current obstacles and potential solutions. Front Nutr 2023; 10:1225233. [PMID: 37743926 PMCID: PMC10513094 DOI: 10.3389/fnut.2023.1225233] [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: 05/18/2023] [Accepted: 08/21/2023] [Indexed: 09/26/2023] Open
Abstract
In vitro meat production presents a potential viable alternative for meat consumption, which could provide the consumer with a product indistinguishable from the original, with very similar nutritional and culinary values. Indeed, the alternative products currently accessible often lack comparable nutritional value or culinary attributes to their animal-derived counterparts. This creates challenges for their global acceptance, particularly in countries where meat consumption holds cultural significance. However, while cultured meat research has been progressing rapidly in recent years, some significant obstacles still need to be overcome before its possible commercialization. Hence, this review summarizes the most current knowledge regarding the history of cultured meat, the currently used cell sources and methods used for the purpose of in vitro meat production, with particular focus on the role of bioreactors, scaffolds and microcarriers in overcoming the current obstacles. The authors put the potential microcarrier and scaffold-based solutions in a context, discussing the ways in which they can impact the way forward for the technology, including the use of considering the potential practical and societal barriers to implementing it as a viable food source worldwide.
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Affiliation(s)
- Magdalena Kulus
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Maurycy Jankowski
- Department of Computer Science and Statistics, Poznan University of Medical Sciences, Poznan, Poland
- Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland
| | - Wiesława Kranc
- Department of Anatomy, Poznan University of Medical Sciences, Poznań, Poland
| | - Afsaneh Golkar Narenji
- Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States
| | - Maryam Farzaneh
- Fertility, Infertility and Perinatology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Piotr Dzięgiel
- Division of Histology and Embryology, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
| | - Maciej Zabel
- Division of Histology and Embryology, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
- Division of Anatomy and Histology, University of Zielona Góra, Zielona Góra, Poland
| | - Paweł Antosik
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Dorota Bukowska
- Department of Basic and Preclinical Sciences, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
| | - Paul Mozdziak
- Prestage Department of Poultry Science, North Carolina State University, Raleigh, NC, United States
- Physiology Graduate Faculty, North Carolina State University, Raleigh, NC, United States
| | - Bartosz Kempisty
- Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland
- Physiology Graduate Faculty, North Carolina State University, Raleigh, NC, United States
- Division of Anatomy, Department of Human Morphology and Embryology, Wroclaw Medical University, Wroclaw, Poland
- Department of Obstetrics and Gynecology, University Hospital and Masaryk University, Brno, Czechia
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16
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Fadl A, Leask A. Hiding in Plain Sight: Human Gingival Fibroblasts as an Essential, Yet Overlooked, Tool in Regenerative Medicine. Cells 2023; 12:2021. [PMID: 37626831 PMCID: PMC10453328 DOI: 10.3390/cells12162021] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2023] [Revised: 08/01/2023] [Accepted: 08/07/2023] [Indexed: 08/27/2023] Open
Abstract
Adult human gingival fibroblasts (HGFs), the most abundant cells in the oral cavity, are essential for maintaining oral homeostasis. Compared with other tissues, adult oral mucosal wounds heal regeneratively, without scarring. Relative to fibroblasts from other locations, HGFs are relatively refractory to myofibroblast differentiation, immunomodulatory, highly regenerative, readily obtained via minimally invasive procedures, easily and rapidly expanded in vitro, and highly responsive to growth factors and cytokines. Consequently, HGFs might be a superior, yet perhaps underappreciated, source of adult mesenchymal progenitor cells to use in tissue engineering and regeneration applications, including the treatment of fibrotic auto-immune connective tissue diseases such as scleroderma. Herein, we highlight in vitro and translational studies that have investigated the regenerative and differentiation potential of HGFs, with the objective of outlining current limitations and inspiring future research that could facilitate translating the regenerative potential of HGFs into the clinic.
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Affiliation(s)
| | - Andrew Leask
- College of Dentistry, University of Saskatchewan, 105 Wiggins Road, Saskatoon, SK S7N 5A2, Canada;
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17
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Lyra-Leite DM, Copley RR, Freeman PP, Pongpamorn P, Shah D, McKenna DE, Lenny B, Pinheiro EA, Weddle CJ, Gharib M, Javed H, Fonoudi H, Sapkota Y, Burridge PW. Nutritional requirements of human induced pluripotent stem cells. Stem Cell Reports 2023; 18:1371-1387. [PMID: 37315525 PMCID: PMC10277817 DOI: 10.1016/j.stemcr.2023.05.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2022] [Revised: 05/05/2023] [Accepted: 05/09/2023] [Indexed: 06/16/2023] Open
Abstract
The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.
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Affiliation(s)
- Davi M Lyra-Leite
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | | | | | - Praeploy Pongpamorn
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Disheet Shah
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | | | - Brian Lenny
- Department of Epidemiology and Cancer Control, St. Jude Children's Hospital, Memphis, TN 38105, USA
| | - Emily A Pinheiro
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Carly J Weddle
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Mennat Gharib
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Hoor Javed
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Hananeh Fonoudi
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Yadav Sapkota
- Department of Epidemiology and Cancer Control, St. Jude Children's Hospital, Memphis, TN 38105, USA
| | - Paul W Burridge
- Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA; Center for Pharmacogenomics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
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18
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Thomaidou AC, Goulielmaki M, Tsintarakis A, Zoumpourlis P, Toya M, Christodoulou I, Zoumpourlis V. miRNA-Guided Regulation of Mesenchymal Stem Cells Derived from the Umbilical Cord: Paving the Way for Stem-Cell Based Regeneration and Therapy. Int J Mol Sci 2023; 24:ijms24119189. [PMID: 37298143 DOI: 10.3390/ijms24119189] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 05/19/2023] [Accepted: 05/21/2023] [Indexed: 06/12/2023] Open
Abstract
The human body is an abundant source of multipotent cells primed with unique properties that can be exploited in a multitude of applications and interventions. Mesenchymal stem cells (MSCs) represent a heterogenous population of undifferentiated cells programmed to self-renew and, depending on their origin, differentiate into distinct lineages. Alongside their proven ability to transmigrate toward inflammation sites, the secretion of various factors that participate in tissue regeneration and their immunoregulatory function render MSCs attractive candidates for use in the cytotherapy of a wide spectrum of diseases and conditions, as well as in different aspects of regenerative medicine. In particular, MSCs that can be found in fetal, perinatal, or neonatal tissues possess additional capabilities, including predominant proliferation potential, increased responsiveness to environmental stimuli, and hypoimmunogenicity. Since microRNA (miRNA)-guided gene regulation governs multiple cellular functions, miRNAs are increasingly being studied in the context of driving the differentiation process of MSCs. In the present review, we explore the mechanisms of miRNA-directed differentiation of MSCs, with a special focus on umbilical cord-derived mesenchymal stem cells (UCMSCs), and we identify the most relevant miRNAs and miRNA sets and signatures. Overall, we discuss the potent exploitations of miRNA-driven multi-lineage differentiation and regulation of UCMSCs in regenerative and therapeutic protocols against a range of diseases and/or injuries that will achieve a meaningful clinical impact through maximizing treatment success rates, while lacking severe adverse events.
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Affiliation(s)
- Arsinoe C Thomaidou
- Laboratory of Clinical Virology, Medical School, University of Crete, 71500 Heraklion, Greece
| | - Maria Goulielmaki
- Cancer Immunology and Immunotherapy Center, Cancer Research Center, Saint Savas Cancer Hospital, 11522 Athens, Greece
| | - Antonis Tsintarakis
- Biomedical Applications Unit, Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), 11635 Athens, Greece
| | - Panagiotis Zoumpourlis
- Biomedical Applications Unit, Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), 11635 Athens, Greece
| | - Marialena Toya
- Biomedical Applications Unit, Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), 11635 Athens, Greece
| | - Ioannis Christodoulou
- Biomedical Applications Unit, Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), 11635 Athens, Greece
| | - Vassilis Zoumpourlis
- Biomedical Applications Unit, Institute of Chemical Biology, National Hellenic Research Foundation (NHRF), 11635 Athens, Greece
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19
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Yang Y, He S, Qi Z, Chai X, Zhao Q, Hu B, Li G, Yu Y. Proliferation toxicity and mechanism of novel mixed bromine/chlorine transformation products of tetrabromobisphenol A on human embryonic stem cell. JOURNAL OF HAZARDOUS MATERIALS 2023; 449:131050. [PMID: 36821903 DOI: 10.1016/j.jhazmat.2023.131050] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/30/2022] [Revised: 01/22/2023] [Accepted: 02/18/2023] [Indexed: 06/18/2023]
Abstract
Mixed bromine/chlorine transformation products of tetrabromobisphenol A (ClyBrxBPAs) are mixed halogenated-type compounds recently identified in electronic waste dismantling sites. There are a lack of toxicity data on these compounds. To study their development toxicity, the proliferation toxicity was investigated using human embryonic stem cells (hESC) exposed to the lowest effective dose of two ClyBrxBPA analogues (2-chloro-2',6-dibromobisphenol A and 2,2'-dichloro-6-monobromobisphenol A). For comparison, tetrabromobisphenol A, 2,2',6-tribromobisphenol A, and bisphenol A were also assessed. It was observed that ClyBrxBPAs inhibited hESCs proliferation in a concentration-dependent manner. The cell bioaccumulation efficiency of ClyBrxBPAs was higher than that of tetrabromobisphenol A. Also, ClyBrxBPAs were more toxic than tetrabromobisphenol A, with 2,2'-dichloro-6-monobromobisphenol A exhibiting the most potent toxicity. Furthermore, flow cytometry and oxidative stress results showed that increased reactive oxygen species raised the degree of apoptosis and reduced DNA synthesis. Metabolomics analysis on the effect of ClyBrxBPAs on metabolic pathway alteration showed that ClyBrxBPAs mainly interfered with four metabolic pathways related to amino acid metabolism and biosynthesis. These results provide an initial perspective on the proliferation toxicity of ClyBrxBPAs, indicating development toxicity in children.
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Affiliation(s)
- Yan Yang
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China; Synergy Innovation Institute of GDUT, Shantou 515041, China
| | - Shiyao He
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Zenghua Qi
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Xuyang Chai
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Qiting Zhao
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Beibei Hu
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Guiying Li
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China
| | - Yingxin Yu
- Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, PR China; Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Key Laboratory of City Cluster Environmental Safety and Green Development, School of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China.
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20
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Tian Z, Yu T, Liu J, Wang T, Higuchi A. Introduction to stem cells. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2023; 199:3-32. [PMID: 37678976 DOI: 10.1016/bs.pmbts.2023.02.012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/17/2023]
Abstract
Stem cells have self-renewal capability and can proliferate and differentiate into a variety of functionally active cells that can serve in various tissues and organs. This review discusses the history, definition, and classification of stem cells. Human pluripotent stem cells (hPSCs) mainly include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Embryonic stem cells are derived from the inner cell mass of the embryo. Induced pluripotent stem cells are derived from reprogramming somatic cells. Pluripotent stem cells have the ability to differentiate into cells derived from all three germ layers (endoderm, mesoderm, and ectoderm). Adult stem cells can be multipotent or unipotent and can produce tissue-specific terminally differentiated cells. Stem cells can be used in cell therapy to replace and regenerate damaged tissues or organs.
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Affiliation(s)
- Zeyu Tian
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Tao Yu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Jun Liu
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Ting Wang
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
| | - Akon Higuchi
- State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China; Department of Chemical and Materials Engineering, National Central University, Jhongli, Taoyuan, Taiwan.
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21
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Extracellular Vesicles and Cellular Ageing. Subcell Biochem 2023; 102:271-311. [PMID: 36600137 DOI: 10.1007/978-3-031-21410-3_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Ageing is a complex process characterized by deteriorated performance at multiple levels, starting from cellular dysfunction to organ degeneration. Stem cell-based therapies aim to administrate stem cells that eventually migrate to the injured site to replenish the damaged tissue and recover tissue functionality. Stem cells can be easily obtained and cultured in vitro, and display several qualities such as self-renewal, differentiation, and immunomodulation that make them suitable candidates for stem cell-based therapies. Current animal studies and clinical trials are being performed to assess the safety and beneficial effects of stem cell engraftments for regenerative medicine in ageing and age-related diseases.Since alterations in cell-cell communication have been associated with the development of pathophysiological processes, new research is focusing on the modulation of the microenvironment. Recent research has highlighted the important role of some microenvironment components that modulate cell-cell communication, thus spreading signals from damaged ageing cells to neighbor healthy cells, thereby promoting systemic ageing. Extracellular vesicles (EVs) are small-rounded vesicles released by almost every cell type. EVs cargo includes several bioactive molecules, such as lipids, proteins, and genetic material. Once internalized by target cells, their specific cargo can induce epigenetic modifications and alter the fate of the recipient cells. Also, EV's content is dependent on the releasing cells, thus, EVs can be used as biomarkers for several diseases. Moreover, EVs have been proposed to be used as cell-free therapies that focus on their administration to slow or even reverse some hallmarks of physiological ageing. It is not surprising that EVs are also under study as next-generation therapies for age-related diseases.
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22
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Castel G, David L. Induction of human trophoblast stem cells. Nat Protoc 2022; 17:2760-2783. [PMID: 36241723 DOI: 10.1038/s41596-022-00744-0] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2021] [Accepted: 06/22/2022] [Indexed: 02/07/2023]
Abstract
Cell reprogramming has allowed unprecedented access to human development, from virtually any genome. However, reprogramming yields pluripotent stem cells that can differentiate into all cells that form a fetus, but not extraembryonic annexes. Therefore, a cellular model allowing study of placental development from a broad genomic repertoire is lacking. Here, we describe an optimized protocol to reprogram somatic cells into human induced trophoblast stem cells (hiTSCs) and convert pluripotent stem cells into human converted TSCs (hcTSCs). This protocol enables much-needed genome-specific placental disease modeling. We also detail extravillous trophoblast and syncytiotrophoblast differentiation protocols from hiTSCs and hcTSCs, a necessary step to validate these cells. In total, this protocol takes 4 months and requires advanced cell culture skills, comparable to those necessary for somatic cell reprogramming into human induced pluripotent stem cells.
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Affiliation(s)
- Gaël Castel
- Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064, Nantes, France
| | - Laurent David
- Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064, Nantes, France.
- Nantes Université, CHU Nantes, INSERM, CNRS, BioCore, Nantes, France.
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23
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Katayama M, Fukuda T, Kaneko T, Nakagawa Y, Tajima A, Naito M, Ohmaki H, Endo D, Asano M, Nagamine T, Nakaya Y, Saito K, Watanabe Y, Tani T, Inoue-Murayama M, Nakajima N, Onuma M. Induced pluripotent stem cells of endangered avian species. Commun Biol 2022; 5:1049. [PMID: 36280684 PMCID: PMC9592614 DOI: 10.1038/s42003-022-03964-y] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2020] [Accepted: 09/08/2022] [Indexed: 11/13/2022] Open
Abstract
The number of endangered avian-related species increase in Japan recently. The application of new technologies, such as induced pluripotent stem cells (iPSCs), may contribute to the recovery of the decreasing numbers of endangered animals and conservation of genetic resources. We established novel iPSCs from three endangered avian species (Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl) with seven reprogramming factors (M3O, Sox2, Klf4, c-Myc, Nanog, Lin28, and Klf2). The iPSCs are pluripotency markers and express pluripotency-related genes and differentiated into three germ layers in vivo and in vitro. These three endangered avian iPSCs displayed different cellular characteristics even though the same reprogramming factors use. Japanese ptarmigan-derived iPSCs have different biological characteristics from those observed in other avian-derived iPSCs. Japanese ptarmigan iPSCs contributed to chimeras part in chicken embryos. To the best of our knowledge, our findings provide the first evidence of the potential value of iPSCs as a resource for endangered avian species conservation. iPSCs from three endangered avian species (including Okinawa rail, Japanese ptarmigan, and Blakiston’s fish owl) are developed and characterized as a potential resource for their conservation.
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Affiliation(s)
- Masafumi Katayama
- grid.140139.e0000 0001 0746 5933Biodiversity Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 Japan
| | - Tomokazu Fukuda
- grid.411792.80000 0001 0018 0409Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551 Japan
| | - Takehito Kaneko
- grid.411792.80000 0001 0018 0409Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551 Japan
| | - Yuki Nakagawa
- grid.411792.80000 0001 0018 0409Graduate School of Science and Engineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551 Japan
| | - Atsushi Tajima
- grid.20515.330000 0001 2369 4728Faculty of Life and Environmental Sciences/T-PIRC, University of Tsukuba, 1-1-1 Ten-noh Dai, Tsukuba, Ibaraki 305-8572 Japan
| | - Mitsuru Naito
- grid.410590.90000 0001 0699 0373National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 Japan
| | - Hitomi Ohmaki
- grid.412658.c0000 0001 0674 6856School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai Midorimachi, Ebetsu, Hokkaido 069-8501 Japan
| | - Daiji Endo
- grid.412658.c0000 0001 0674 6856School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai Midorimachi, Ebetsu, Hokkaido 069-8501 Japan
| | - Makoto Asano
- grid.256342.40000 0004 0370 4927Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193 Japan
| | - Takashi Nagamine
- Okinawa Wildlife Federation, 308-7-205 Maehara, Uruma, Okinawa 904-2235 Japan
| | - Yumiko Nakaya
- Okinawa Wildlife Federation, 308-7-205 Maehara, Uruma, Okinawa 904-2235 Japan
| | - Keisuke Saito
- Institute for Raptor Biomedicine Japan (Kushiro Shitsugen Wildlife Center), 2-2101 Hokuto, Kushiro, Hokkaido 084-0922 Japan
| | - Yukiko Watanabe
- Institute for Raptor Biomedicine Japan (Kushiro Shitsugen Wildlife Center), 2-2101 Hokuto, Kushiro, Hokkaido 084-0922 Japan
| | - Tetsuya Tani
- grid.258622.90000 0004 1936 9967Department of Agriculture, Kindai University, 3327-204 Nakamachi, Nara, 631-0052 Japan
| | - Miho Inoue-Murayama
- grid.258799.80000 0004 0372 2033Wildlife Research Center, Kyoto University, 2-24 Tanaka-Sekiden-Cho, Sakyo-Ku, Kyoto 606-8203 Japan
| | - Nobuyoshi Nakajima
- grid.140139.e0000 0001 0746 5933Biodiversity Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 Japan
| | - Manabu Onuma
- grid.140139.e0000 0001 0746 5933Biodiversity Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 Japan ,grid.412658.c0000 0001 0674 6856School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai Midorimachi, Ebetsu, Hokkaido 069-8501 Japan
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24
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The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells. J Clin Med 2022; 11:jcm11206087. [PMID: 36294410 PMCID: PMC9605397 DOI: 10.3390/jcm11206087] [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: 08/25/2022] [Revised: 09/30/2022] [Accepted: 10/07/2022] [Indexed: 11/06/2022] Open
Abstract
Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.
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25
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Xu X, Feng Q, Ma X, Deng Y, Zhang K, Ooi HS, Yang B, Zhang ZY, Feng B, Bian L. Dynamic gelatin-based hydrogels promote the proliferation and self-renewal of embryonic stem cells in long-term 3D culture. Biomaterials 2022; 289:121802. [PMID: 36152514 DOI: 10.1016/j.biomaterials.2022.121802] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2022] [Revised: 08/12/2022] [Accepted: 09/09/2022] [Indexed: 11/02/2022]
Abstract
Long-term maintenance of embryonic stem cells (ESCs) in the undifferentiated state is still challenging. Compared with traditional 2D culture methods, 3D culture in biomaterials such as hydrogels is expected to better support the long-term self-renewal of ESCs by emulating the biophysical and biochemical properties of the extracellular matrix (ECM). Although prior studies showed that soft and degradable hydrogels favor the 3D growth of ESCs, few studies have examined the impact of the structural dynamics of the hydrogel matrix on ESC behaviors. Herein, we report a gelatin-based structurally dynamic hydrogel (GelCD hydrogel) that emulates the intrinsic structural dynamics of the ECM. Compared with covalently crosslinked gelatin hydrogels (GelMA hydrogels) with similar stiffness and biodegradability, GelCD hydrogels significantly promote the clonal expansion and viability of encapsulated mouse ESCs (mESCs) independent of MMP-mediated hydrogel degradation. Furthermore, GelCD hydrogels better maintain the pluripotency of encapsulated mESCs than do traditional 2D culture methods that use MEF feeder cells or medium supplementation with GSK3β and MEK 1/2 inhibitors (2i). When cultured in GelCD hydrogels for an extended period (over 2 months) with cell passaging every 7 days, mESCs preserve their normal morphology and maintain their pluripotency and full differentiation capability. Our findings highlight the critical role of the structural dynamics of the hydrogel matrix in accommodating the volume expansion that occurs during clonal ESC growth, and we believe that our dynamic hydrogels represent a valuable tool to support the long-term 3D culture of ESCs.
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Affiliation(s)
- Xiayi Xu
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China.
| | - Qian Feng
- Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing, 400044, China; Chongqing Key Laboratory of Soft-Matter Material Chemistry and Function Manufacturing, Chongqing, 400044, China
| | - Xun Ma
- Center for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Chinese Academy of Sciences Limited, Hong Kong SAR, 999077, China; School of Biomedical Sciences, Faculty of Medicine, Institute for Tissue Engineering and Regenerative Medicine (iTERM), CUHK-GIBH Joint Research Laboratory on Stem Cells and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China
| | - Yingrui Deng
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China
| | - Kunyu Zhang
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China; School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou, 511442, China
| | - Hon Son Ooi
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China
| | - Boguang Yang
- Department of Biomedical Engineering, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China
| | - Zhi-Yong Zhang
- Translational Research Centre of Regenerative Medicine and 3D Printing of Guangzhou Medical University, Guangdong Province Engineering Research Center for Biomedical Engineering, State Key Laboratory of Respiratory Disease, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou City, Guangdong Province, 510150, China.
| | - Bo Feng
- Center for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Chinese Academy of Sciences Limited, Hong Kong SAR, 999077, China; School of Biomedical Sciences, Faculty of Medicine, Institute for Tissue Engineering and Regenerative Medicine (iTERM), CUHK-GIBH Joint Research Laboratory on Stem Cells and Regenerative Medicine, The Chinese University of Hong Kong, Hong Kong SAR, 999077, China; Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China.
| | - Liming Bian
- School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou, 511442, China; National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou, 510006, China; Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South China University of Technology, Guangzhou, 510006, China; Guangdong Provincial Key Laboratory of Biomedical Engineering, South China University of Technology, Guangzhou, 510006, China.
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26
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Epigenetic Alterations under Oxidative Stress in Stem Cells. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2022; 2022:6439097. [PMID: 36071870 PMCID: PMC9444469 DOI: 10.1155/2022/6439097] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/15/2022] [Revised: 07/16/2022] [Accepted: 07/27/2022] [Indexed: 11/18/2022]
Abstract
Epigenetic regulation of gene expression, including DNA methylation and histone modifications, provides finely tuned responses for cells that undergo cellular environment changes. Abundant evidences have demonstrated the detrimental role of oxidative stress in various human pathogenesis since oxidative stress results from the imbalance between reactive oxygen species (ROS) accumulation and antioxidant defense system. Stem cells can self-renew themselves and meanwhile have the potential to differentiate into many other cell types. As some studies have described the effects of oxidative stress on homeostasis and cell fate decision of stem cells, epigenetic alterations have emerged crucial for mediating the stem cell behaviours under oxidative stress. Here, we review recent findings on the oxidative effects on DNA and histone modifications in stem cells. We propose that epigenetic alterations and oxidative stress may influence each other in stem cells.
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27
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Linkova DD, Rubtsova YP, Egorikhina MN. Cryostorage of Mesenchymal Stem Cells and Biomedical Cell-Based Products. Cells 2022; 11:cells11172691. [PMID: 36078098 PMCID: PMC9454587 DOI: 10.3390/cells11172691] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 08/15/2022] [Accepted: 08/24/2022] [Indexed: 11/16/2022] Open
Abstract
Mesenchymal stem cells (MSCs) manifest vast opportunities for clinical use due both to their ability for self-renewal and for effecting paracrine therapeutic benefits. At the same time, difficulties with non-recurrent generation of large numbers of cells due to the necessity for long-term MSC expansion ex vivo, or the requirement for repeated sampling of biological material from a patient significantly limits the current use of MSCs in clinical practice. One solution to these problems entails the creation of a biobank using cell cryopreservation technology. This review is aimed at analyzing and classifying literature data related to the development of protocols for the cryopreservation of various types of MSCs and tissue-engineered structures. The materials in the review show that the existing techniques and protocols for MSC cryopreservation are very diverse, which significantly complicates standardization of the entire process. Here, the selection of cryoprotectors and of cryoprotective media shows the greatest variability. Currently, it is the cryopreservation of cell suspensions that has been studied most extensively, whereas there are very few studies in the literature on the freezing of intact tissues or of tissue-engineered structures. However, even now it is possible to develop general recommendations to optimize the cryopreservation process, making it less traumatic for cells.
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28
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Chen G, Yin S, Zeng H, Li H, Wan X. Regulation of Embryonic Stem Cell Self-Renewal. LIFE (BASEL, SWITZERLAND) 2022; 12:life12081151. [PMID: 36013330 PMCID: PMC9410528 DOI: 10.3390/life12081151] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 07/12/2022] [Accepted: 07/21/2022] [Indexed: 11/16/2022]
Abstract
Embryonic stem cells (ESCs) are a type of cells capable of self-renewal and multi-directional differentiation. The self-renewal of ESCs is regulated by factors including signaling pathway proteins, transcription factors, epigenetic regulators, cytokines, and small molecular compounds. Similarly, non-coding RNAs, small RNAs, and microRNAs (miRNAs) also play an important role in the process. Functionally, the core transcription factors interact with helper transcription factors to activate the expression of genes that contribute to maintaining pluripotency, while suppressing the expression of differentiation-related genes. Additionally, cytokines such as leukemia suppressor factor (LIF) stimulate downstream signaling pathways and promote self-renewal of ESCs. Particularly, LIF binds to its receptor (LIFR/gp130) to trigger the downstream Jak-Stat3 signaling pathway. BMP4 activates the downstream pathway and acts in combination with Jak-Stat3 to promote pluripotency of ESCs in the absence of serum. In addition, activation of the Wnt-FDZ signaling pathway has been observed to facilitate the self-renewal of ESCs. Small molecule modulator proteins of the pathway mentioned above are widely used in in vitro culture of stem cells. Multiple epigenetic regulators are involved in the maintenance of ESCs self-renewal, making the epigenetic status of ESCs a crucial factor in this process. Similarly, non-coding RNAs and cellular energetics have been described to promote the maintenance of the ESC's self-renewal. These factors regulate the self-renewal and differentiation of ESCs by forming signaling networks. This review focused on the role of major transcription factors, signaling pathways, small molecular compounds, epigenetic regulators, non-coding RNAs, and cellular energetics in ESC's self-renewal.
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Affiliation(s)
- Guofang Chen
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
| | - Shasha Yin
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
| | - Hongliang Zeng
- Institute of Chinese Materia Medica, Hunan Academy of Chinese Medicine, Changsha 410013, China;
| | - Haisen Li
- School of Medicine, Wayne State University, Detroit, MI 48201, USA
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
| | - Xiaoping Wan
- Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, Shanghai 201204, China;
- Correspondence: (G.C.); (H.L.); (X.W.); Tel./Fax: +86-021-20261000 (ext. 1379) (G.C.)
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Alsobaie S, Alsobaie T, Mantalaris S. Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells. STEM CELLS AND CLONING: ADVANCES AND APPLICATIONS 2022; 15:43-52. [PMID: 35812359 PMCID: PMC9259205 DOI: 10.2147/sccaa.s365776] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/10/2022] [Accepted: 06/22/2022] [Indexed: 01/16/2023]
Abstract
Introduction High-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application. Methods To address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells. Results Through immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities. Discussion We showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.
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Affiliation(s)
- Sarah Alsobaie
- Department of Clinical Laboratory Science, King Saud University, Riyadh, 11451, Saudi Arabia
- Correspondence: Sarah Alsobaie, Department of Clinical Laboratory Science, King Saud University, Prince Turki Alawal Street, Riyadh, 11451, Saudi Arabia, Tel +966 507191011, Fax +966 114677580, Email
| | - Tamador Alsobaie
- Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, SW7 2AZ, UK
| | - Sakis Mantalaris
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30322, USA
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Lezmi E, Benvenisty N. The Tumorigenic Potential of Human Pluripotent Stem Cells. Stem Cells Transl Med 2022; 11:791-796. [PMID: 35679163 PMCID: PMC9397652 DOI: 10.1093/stcltm/szac039] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 04/24/2022] [Indexed: 11/23/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are currently evaluated for clinical applications due to their proliferation and differentiation capacities, raising the need to both assess and enhance, the safety of hPSC-based treatments. Distinct molecular features contribute to the tumorigenicity of hPSCs, manifested in the formation of teratoma tumors upon transplantation in vivo. Prolonged in vitro culturing of hPSCs can enhance selection for specific genetic aberrations, either at the chromosome or gene level. Some of these aberrations are tightly linked to human tumor pathology and increase the tumorigenic aggressiveness of the abnormal cells. In this perspective, we describe major tumor-associated risk factors entailed in hPSC-based therapy, and present precautionary and safety measures relevant for the development and application of such therapies.
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Affiliation(s)
- Elyad Lezmi
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel
| | - Nissim Benvenisty
- The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel
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31
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Development of an efficient single-cell cloning and expansion strategy for genome edited induced pluripotent stem cells. Mol Biol Rep 2022; 49:7887-7898. [PMID: 35637316 DOI: 10.1007/s11033-022-07621-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 05/19/2022] [Indexed: 01/31/2023]
Abstract
BACKGROUND Disease-specific human induced pluripotent stem cells (hiPSCs) can be generated directly from individuals with known disease characteristics or alternatively be modified using genome editing approaches to introduce disease causing genetic mutations to study the biological response of those mutations. The genome editing procedure in hiPSCs is still inefficient, particularly when it comes to homology directed repair (HDR) of genetic mutations or targeted transgene insertion in the genome and single cell cloning of edited cells. In addition, genome editing processes also involve additional cellular stresses such as poor cell viability and genetic stability of hiPSCs. Therefore, efficient workflows are desired to increase genome editing application to hiPSC disease models and therapeutic applications. METHODS AND RESULTS To this end, we demonstrate an efficient workflow for feeder-free single cell clone generation and expansion in both CRISPR-mediated knock-out (KO) and knock-in (KI) hiPSC lines. Using StemFlex medium and CloneR supplement in conjunction with Matrigel cell culture matrix, we show that cell viability and expansion during single-cell cloning in edited and unedited cells is significantly enhanced. Keeping all factors into account, we have successfully achieved hiPSC single-cell survival and cloning in both edited and unedited cells with rates as maximum as 70% in less than 2 weeks. CONCLUSION This simplified and efficient workflow will allow for a new level of sophistication in generating hiPSC-based disease models to promote rapid advancement in basic research and also the development of novel cellular therapeutics.
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32
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Truong RD, Bernier MA, Dennis JE, Kean TJ. Synoviocyte-Derived Extracellular Matrix and bFGF Speed Human Chondrocyte Proliferation While Maintaining Differentiation Potential. Front Bioeng Biotechnol 2022; 10:825005. [PMID: 35685088 PMCID: PMC9171110 DOI: 10.3389/fbioe.2022.825005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 04/15/2022] [Indexed: 11/25/2022] Open
Abstract
Improving the ability of human chondrocytes to proliferate, while maintaining their differentiation potential, has presented a great challenge in cartilage tissue engineering. In this study, human chondrocytes were cultured under four unique growth conditions at physiologic oxygen tension: tissue culture plastic (TCP) only, synoviocyte matrix (SCM)-coated flasks only, SCM-coated flasks with bFGF media supplement, and TCP with bFGF media supplement. The results indicated that, compared to standard TCP, all test conditions showed significantly increased cell expansion rates and an increase in both glycosaminoglycan (GAG) and collagen content during redifferentiation culture. Specifically, the combined SCM + bFGF growth condition showed an additive effect, with an increase of approximately 36% more cells per passage (5-7 days) when compared to the SCM alone. In conclusion, the results of this study demonstrate that bFGF and SCM can be used as supplements to enhance the growth of human chondrocytes both as individual enhancers and as a combined additive.
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Affiliation(s)
- Rachel D. Truong
- College of Medicine, University of Central Florida, Orlando, FL, United States
| | - Megan A. Bernier
- College of Medicine, University of Central Florida, Orlando, FL, United States
| | - James E. Dennis
- Department of Orthopedic Surgery, Baylor College of Medicine, Houston, TX, United States
| | - Thomas J. Kean
- College of Medicine, University of Central Florida, Orlando, FL, United States
- Department of Orthopedic Surgery, Baylor College of Medicine, Houston, TX, United States
- Biionix Cluster, Internal Medicine, College of Medicine, University of Central Florida, Orlando, FL, United States
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Abstract
Although tumourigenesis occurs due to genetic mutations, the role of epigenetic dysregulations in cancer is also well established. Epigenetic dysregulations in cancer may occur as a result of mutations in genes encoding histone/DNA-modifying enzymes and chromatin remodellers or mutations in histone protein itself. It is also true that misregulated gene expression without genetic mutations in these factors could also support tumour initiation and progression. Interestingly, metabolic rewiring has emerged as a hallmark of cancer due to gene mutations in specific metabolic enzymes or dietary/environmental factors. Recent studies report an intricate cross-talk between epigenetic and metabolic reprogramming in cancer. This review discusses the role of epigenetic and metabolic dysregulations and their cross-talk in tumourigenesis with a special focus on gliomagenesis. We also discuss the role of recently developed human embryonic stem cells/induced pluripotent stem cells-derived organoid models of gliomas and how these models are proving instrumental in uncovering human-specific cellular and molecular complexities of gliomagenesis.
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Affiliation(s)
- Bismi Phasaludeen
- Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates
| | - Bright Starling Emerald
- Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates,Zayed Center for Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates
| | - Suraiya Anjum Ansari
- Department of Biochemistry and Molecular Biology, College of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, Abu Dhabi, United Arab Emirates,Zayed Center for Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates
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Aydin O, Passaro AP, Raman R, Spellicy SE, Weinberg RP, Kamm RD, Sample M, Truskey GA, Zartman J, Dar RD, Palacios S, Wang J, Tordoff J, Montserrat N, Bashir R, Saif MTA, Weiss R. Principles for the design of multicellular engineered living systems. APL Bioeng 2022; 6:010903. [PMID: 35274072 PMCID: PMC8893975 DOI: 10.1063/5.0076635] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Accepted: 02/02/2022] [Indexed: 12/14/2022] Open
Abstract
Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the "black box" of living cells.
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Affiliation(s)
| | - Austin P. Passaro
- Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30602, USA
| | - Ritu Raman
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
| | | | - Robert P. Weinberg
- School of Pharmacy, Massachusetts College of Pharmacy and Health Sciences, Boston, Massachusetts 02115, USA
| | | | - Matthew Sample
- Center for Ethics and Law in the Life Sciences, Leibniz Universität Hannover, 30167 Hannover, Germany
| | - George A. Truskey
- Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708, USA
| | - Jeremiah Zartman
- Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, USA
| | - Roy D. Dar
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
| | - Sebastian Palacios
- Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139, USA
| | - Jason Wang
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
| | - Jesse Tordoff
- Computational and Systems Biology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
| | - Nuria Montserrat
- Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 08028 Barcelona, Spain
| | | | - M. Taher A. Saif
- Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA
| | - Ron Weiss
- Author to whom correspondence should be addressed:
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Zhou P, Qin L, Ge Z, Xie B, Huang H, He F, Ma S, Ren L, Shi J, Pei S, Dong G, Qi Y, Lan F. Design of chemically defined synthetic substrate surfaces for the in vitro maintenance of human pluripotent stem cells: A review. J Biomed Mater Res B Appl Biomater 2022; 110:1968-1990. [PMID: 35226397 DOI: 10.1002/jbm.b.35034] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 01/10/2022] [Accepted: 01/17/2022] [Indexed: 11/11/2022]
Abstract
Human pluripotent stem cells (hPSCs) have the potential of long-term self-renewal and differentiation into nearly all cell types in vitro. Prior to the downstream applications, the design of chemically defined synthetic substrates for the large-scale proliferation of quality-controlled hPSCs is critical. Although great achievements have been made, Matrigel and recombinant proteins are still widely used in the fundamental research and clinical applications. Therefore, much effort is still needed to improve the performance of synthetic substrates in the culture of hPSCs, realizing their commercial applications. In this review, we summarized the design of reported synthetic substrates and especially their limitations in terms of cell culture. Moreover, much attention was paid to the development of promising peptide displaying surfaces. Besides, the biophysical regulation of synthetic substrate surfaces as well as the three-dimensional culture systems were described.
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Affiliation(s)
- Ping Zhou
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Liying Qin
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Zhangjie Ge
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Biyao Xie
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Hongxin Huang
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Fei He
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Shengqin Ma
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Lina Ren
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Jiamin Shi
- Department of Laboratory Animal Centre, Changzhi Medical College, Changzhi, China
| | - Suying Pei
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Genxi Dong
- School of Stomatology, Lanzhou University, Lanzhou, China
| | - Yongmei Qi
- School of Life Sciences, Lanzhou University, Lanzhou, China
| | - Feng Lan
- Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen Key Laboratory of Cardiovascular Disease, State Key Laboratory of Cardiovascular Disease, Shenzhen, China
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Cell Surface Proteins for Enrichment and In Vitro Characterization of Human Pluripotent Stem Cell-Derived Myogenic Progenitors. Stem Cells Int 2022; 2022:2735414. [PMID: 35251185 PMCID: PMC8894063 DOI: 10.1155/2022/2735414] [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] [Received: 10/15/2021] [Revised: 01/04/2022] [Accepted: 01/12/2022] [Indexed: 11/17/2022] Open
Abstract
Human myogenic progenitors can be derived from pluripotent stem cells (PSCs) for use in modeling natural and pathological myogenesis, as well as treating muscle diseases. Transgene-free methods of deriving myogenic progenitors from different PSC lines often produce mixed populations that are heterogeneous in myogenic differentiation potential, yet detailed and accurate characterization of human PSC-derived myogenic progenitors remains elusive in the field. The isolation and purification of human PSC-derived myogenic progenitors is thus an important methodological consideration when we investigate the properties and behaviors of these cells in culture. We previously reported a transgene-free, serum-free floating sphere culture method for the derivation of myogenic progenitors from human PSCs. In this study, we first performed comprehensive cell surface protein profiling of the sphere culture cells through the screening of 255 antibodies. Next, we used magnetic activated cell sorting and enriched the cells according to the expression of specific surface markers. The ability of muscle differentiation in the resulting cells was characterized by immunofluorescent labeling and quantification of positively stained cells. Our results revealed that myotube-forming cells resided in the differentiated cultures of CD29+, CD56+, CD271+, and CD15– fractions, while thick and multinucleated myotubes were identified in the differentiated cultures from CD9+ and CD146+ fractions. We found that PAX7 localization to the nucleus correlates with myotube-forming ability in these sorted populations. We also demonstrated that cells in unsorted, CD271+, and CD15– fractions responded differently to cryopreservation and prolonged culture expansion. Lastly, we showed that CD271 expression is essential for terminal differentiation of human PSC-derived myogenic progenitors. Taken together, these cell surface proteins are not only useful markers to identify unique cellular populations in human PSC-derived myogenic progenitors but also functionally important molecules that can provide valuable insight into human myogenesis.
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37
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Effects of embryonic stem cell-conditioned medium on the preimplantation development of mouse embryos. ZYGOTE 2022; 30:464-470. [PMID: 35172909 DOI: 10.1017/s0967199421000575] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The production of high-quality embryos in the laboratory and a successful pregnancy are closely related to the condition and contents of oocyte and embryo culture media. In this study, we investigated the effects of embryonic stem cell-conditioned medium (ESCCM) and embryonic stem cells growth medium (ESCGM) compared with potassium-enriched simplex optimized medium (KSOM) on preimplantation embryo development stages during natural or in vitro fertilization (IVF). Birth rate of pups was measured. To obtain mature oocytes, and 2-cell and 8-cell embryos, human chorionic gonadotropin (HCG) was injected 48 h after i.p. injection of 5 units of pregnant mare serum gonadotropin. Mature oocytes were obtained from non-mated female mice 14 h after HCG injection. To obtain 2-cell and 8-cell embryos, mated female mice, 1 day and 3 days, respectively, after HCG injection, were used. Mature oocytes were fertilized in HTF medium. Embryos obtained from natural or in vitro fertilization were cultured in experimental media, ESCCM and ESCGM, or KSOM as the control culture medium. Embryos that developed to the blastocyst stage were transferred to the uteri of pseudopregnant mice and effects of the experimental media on embryo viability were determined. ESCCM and ESCGM could not pass the embryo after the 2-cell stage, but they were suitable for the development of the embryo from the 8-cell stage to the blastocyst. It can be concluded that the embryo has various requirements at different stages of development.
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38
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Histone modifications in neurodifferentiation of embryonic stem cells. Heliyon 2022; 8:e08664. [PMID: 35028451 PMCID: PMC8741459 DOI: 10.1016/j.heliyon.2021.e08664] [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: 09/30/2021] [Revised: 11/25/2021] [Accepted: 12/21/2021] [Indexed: 11/30/2022] Open
Abstract
Post-translational modifications of histone proteins regulate a long cascade of downstream cellular activities, including transcription and replication. Cellular lineage differentiation involves large-scale intracellular signaling and extracellular context. In particular, histone modifications play instructive and programmatic roles in central nervous system development. Deciphering functions of histone could offer feasible molecular strategies for neural diseases caused by histone modifications. Here, we review recent advances of in vitro and in vivo studies on histone modifications in neural differentiation.
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39
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Chen L, Guttieres D, Koenigsberg A, Barone PW, Sinskey AJ, Springs SL. Large-scale cultured meat production: Trends, challenges and promising biomanufacturing technologies. Biomaterials 2021; 280:121274. [PMID: 34871881 DOI: 10.1016/j.biomaterials.2021.121274] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2021] [Revised: 11/17/2021] [Accepted: 11/23/2021] [Indexed: 02/07/2023]
Abstract
Food systems of the future will need to face an increasingly clear reality - that a protein-rich diet is essential for good health, but traditional meat products will not suffice to ensure safety, sustainability, and equity of food supply chains at a global scale. This paper provides an in-depth analysis of bioprocess technologies needed for cell-based meat production and challenges in reaching commercial scale. Specifically, it reviews state-of-the-art bioprocess technologies, current limitations, and opportunities for research across four domains: cell line development, cell culture media, scaffolding, and bioreactors. This also includes exploring innovations to make cultured meat a viable protein alternative across numerous key performance indicators and for specific applications where traditional livestock is not an option (e.g., local production, space exploration). The paper explores tradeoffs between production scale, product quality, production cost, and footprint over different time horizons. Finally, a discussion explores various factors that may impact the ability to successfully scale and market cultured meat products: social acceptance, environmental tradeoffs, regulatory guidance, and public health benefits. While the exact nature of the transition from traditional livestock to alternative protein products is uncertain, it has already started and will likely continue to build momentum in the next decade.
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Affiliation(s)
- Lu Chen
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States
| | - Donovan Guttieres
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States
| | - Andrea Koenigsberg
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States
| | - Paul W Barone
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States
| | - Anthony J Sinskey
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States
| | - Stacy L Springs
- Massachusetts Institute of Technology, Center for Biomedical Innovation, Cambridge, MA, United States.
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40
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Human Induced Pluripotent Stem Cell-Derived Vascular Cells: Recent Progress and Future Directions. J Cardiovasc Dev Dis 2021; 8:jcdd8110148. [PMID: 34821701 PMCID: PMC8622843 DOI: 10.3390/jcdd8110148] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 11/01/2021] [Accepted: 11/02/2021] [Indexed: 12/12/2022] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) hold great promise for cardiovascular regeneration following ischemic injury. Considerable effort has been made toward the development and optimization of methods to differentiate hiPSCs into vascular cells, such as endothelial and smooth muscle cells (ECs and SMCs). In particular, hiPSC-derived ECs have shown robust potential for promoting neovascularization in animal models of cardiovascular diseases, potentially achieving significant and sustained therapeutic benefits. However, the use of hiPSC-derived SMCs that possess high therapeutic relevance is a relatively new area of investigation, still in the earlier investigational stages. In this review, we first discuss different methodologies to derive vascular cells from hiPSCs with a particular emphasis on the role of key developmental signals. Furthermore, we propose a standardized framework for assessing and defining the EC and SMC identity that might be suitable for inducing tissue repair and regeneration. We then highlight the regenerative effects of hiPSC-derived vascular cells on animal models of myocardial infarction and hindlimb ischemia. Finally, we address several obstacles that need to be overcome to fully implement the use of hiPSC-derived vascular cells for clinical application.
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41
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The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells. Int J Mol Sci 2021; 22:ijms222111418. [PMID: 34768848 PMCID: PMC8584070 DOI: 10.3390/ijms222111418] [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] [Received: 08/22/2021] [Revised: 10/05/2021] [Accepted: 10/20/2021] [Indexed: 12/02/2022] Open
Abstract
Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell’s morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.
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42
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Building Pluripotency Identity in the Early Embryo and Derived Stem Cells. Cells 2021; 10:cells10082049. [PMID: 34440818 PMCID: PMC8391114 DOI: 10.3390/cells10082049] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Revised: 07/27/2021] [Accepted: 08/06/2021] [Indexed: 12/13/2022] Open
Abstract
The fusion of two highly differentiated cells, an oocyte with a spermatozoon, gives rise to the zygote, a single totipotent cell, which has the capability to develop into a complete, fully functional organism. Then, as development proceeds, a series of programmed cell divisions occur whereby the arising cells progressively acquire their own cellular and molecular identity, and totipotency narrows until when pluripotency is achieved. The path towards pluripotency involves transcriptome modulation, remodeling of the chromatin epigenetic landscape to which external modulators contribute. Both human and mouse embryos are a source of different types of pluripotent stem cells whose characteristics can be captured and maintained in vitro. The main aim of this review is to address the cellular properties and the molecular signature of the emerging cells during mouse and human early development, highlighting similarities and differences between the two species and between the embryos and their cognate stem cells.
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Capparè P, Tetè G, Sberna MT, Panina-Bordignon P. The Emerging Role of Stem Cells in Regenerative Dentistry. Curr Gene Ther 2021; 20:259-268. [PMID: 32811413 DOI: 10.2174/1566523220999200818115803] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2020] [Revised: 07/25/2020] [Accepted: 07/29/2020] [Indexed: 02/06/2023]
Abstract
Progress of modern dentistry is accelerating at a spectacular speed in the scientific, technological and clinical areas. Practical examples are the advancement in the digital field, which has guaranteed an average level of prosthetic practices for all patients, as well as other scientific developments, including research on stem cell biology. Given their plasticity, defined as the ability to differentiate into specific cell lineages with a capacity of almost unlimited self-renewal and release of trophic/immunomodulatory factors, stem cells have gained significant scientific and commercial interest in the last 15 years. Stem cells that can be isolated from various tissues of the oral cavity have emerged as attractive sources for bone and dental regeneration, mainly due to their ease of accessibility. This review will present the current understanding of emerging conceptual and technological issues of the use of stem cells to treat bone and dental loss defects. In particular, we will focus on the clinical application of stem cells, either directly isolated from oral sources or in vitro reprogrammed from somatic cells (induced pluripotent stem cells). Research aimed at further unraveling stem cell plasticity will allow to identify optimal stem cell sources and characteristics, to develop novel regenerative tools in dentistry.
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Affiliation(s)
- Paolo Capparè
- Department of Dentistry, IRCCS San Raffaele Hospital, Milan, Italy,Dental School, Vita-Salute San Raffaele University, School of Medicine, Milan, Italy
| | - Giulia Tetè
- Department of Dentistry, IRCCS San Raffaele Hospital, Milan, Italy
| | | | - Paola Panina-Bordignon
- Neuroimmunology Unit, Institute of Experimental Neurology, IRCCS San Raffaele Hospital, Milan, Italy,Dental School, Vita-Salute San Raffaele University, School of Medicine, Milan, Italy
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Kronstein-Wiedemann R, Thiel J, Tonn T. Blood Pharming – eine realistische Option? TRANSFUSIONSMEDIZIN 2021. [DOI: 10.1055/a-1342-0820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
ZusammenfassungDie Bluttransfusion ist ein wesentlicher und unersetzlicher Teil der modernen Medizin. Jedoch stellt vor allem bei Patienten mit sehr seltenen Blutgruppenkonstellationen der Mangel an Blutprodukten auch heute noch ein wichtiges Gesundheitsproblem weltweit dar. Um diesem Problem entgegenzutreten, versucht man seit einiger Zeit künstlich rote Blutzellen zu generieren. Diese haben potenzielle Vorteile gegenüber Spenderblut, wie z. B. ein verringertes Risiko für die Übertragung von Infektionskrankheiten. Diese Übersicht fasst die aktuellen Entwicklungen über den Prozess der Erythropoese, die Expansionsstrategien der erythrozytären Zellen, der verschiedenen Quellen für ex vivo expandierte Erythrozyten, die Hürden für die klinische Anwendung und die zukünftigen Möglichkeiten der Anwendung zusammen.
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Affiliation(s)
- Romy Kronstein-Wiedemann
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
| | - Jessica Thiel
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
| | - Torsten Tonn
- DRK-Blutspendedienst Nord-Ost gGmbH/Institut Dresden
- Experimentelle Transfusionsmedizin, Medizinische Fakultät Universitätsklinikum Carl Gustav Carus
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Abstract
The cultured meat market has been growing at an accelerated space since the first creation of cultured meat burger back in 2013. Substantial efforts have been made to reduce costs by eliminating serum in growth media and improving process efficiency by employing bioreactors. In parallel, efforts are also being made on scaffolding innovations to offer better cells proliferation, differentiation and tissue development. So far, scaffolds used in cultured meat research are predominantly collagen and gelatin, which are animal-derived. To align with cell-based meat vision i.e. environment conservation and animal welfare, plant-derived biomaterials for scaffolding are being intensively explored. This paper reviews and discusses the advantages and disadvantages of scaffold materials and potential scaffolding related to scale-up solution for the production of cultured meat.
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Affiliation(s)
- Jasmine Si Han Seah
- School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore
| | - Satnam Singh
- Biomanufacturing Technology, Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
| | - Lay Poh Tan
- School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore
| | - Deepak Choudhury
- Biomanufacturing Technology, Bioprocessing Technology Institute (BTI), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
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Pavarajarn W, Rungsiwiwut R, Numchaisrika P, Virutamasen P, Pruksananonda K. Human Caesarean scar-derived feeder cells: a novel feeder cell type for culturing human pluripotent stem cells without exogenous basic fibroblast growth factor supplementation. Reprod Fertil Dev 2021; 32:822-834. [PMID: 32527373 DOI: 10.1071/rd19128] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2019] [Accepted: 12/10/2019] [Indexed: 11/23/2022] Open
Abstract
In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-β1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.
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Affiliation(s)
- Wipawee Pavarajarn
- Graduate School, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand; and Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand
| | - Ruttachuk Rungsiwiwut
- Department of Anatomy, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, Thailand
| | - Pranee Numchaisrika
- Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand
| | - Pramuan Virutamasen
- Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand
| | - Kamthorn Pruksananonda
- Human Embryonic Stem Cell Research Center, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4, Bangkok 10330, Thailand; and Corresponding author.
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Eibl R, Senn Y, Gubser G, Jossen V, van den Bos C, Eibl D. Cellular Agriculture: Opportunities and Challenges. Annu Rev Food Sci Technol 2021; 12:51-73. [PMID: 33770467 DOI: 10.1146/annurev-food-063020-123940] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Cellular agriculture is the controlled and sustainable manufacture of agricultural products with cells and tissues without plant or animal involvement. Today, microorganisms cultivated in bioreactors already produce egg and milk proteins, sweeteners, and flavors for human nutrition as well as leather and fibers for shoes, bags, and textiles. Furthermore, plant cell and tissue cultures provide ingredients that stimulate the immune system and improve skin texture, with another precommercial cellular agriculture product, in vitro meat, currently receiving a great deal of attention. All these approaches could assist traditional agriculture in continuing to provide for the dietary requirements of a growing world population while freeing up important resources such as arable land. Despite early successes, challenges remain and are discussed in this review, with a focus on production processes involving plant and animal cell and tissue cultures.
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Affiliation(s)
- Regine Eibl
- Institute of Chemistry and Biotechnology, Department of Life Sciences and Facility Management, Zurich University of Applied Sciences, Wädenswil 8820, Switzerland;
| | - Yannick Senn
- Institute of Chemistry and Biotechnology, Department of Life Sciences and Facility Management, Zurich University of Applied Sciences, Wädenswil 8820, Switzerland;
| | - Géraldine Gubser
- Institute of Chemistry and Biotechnology, Department of Life Sciences and Facility Management, Zurich University of Applied Sciences, Wädenswil 8820, Switzerland;
| | - Valentin Jossen
- Institute of Chemistry and Biotechnology, Department of Life Sciences and Facility Management, Zurich University of Applied Sciences, Wädenswil 8820, Switzerland;
| | | | - Dieter Eibl
- Institute of Chemistry and Biotechnology, Department of Life Sciences and Facility Management, Zurich University of Applied Sciences, Wädenswil 8820, Switzerland;
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Sarkar A, Saha S, Paul A, Maji A, Roy P, Maity TK. Understanding stem cells and its pivotal role in regenerative medicine. Life Sci 2021; 273:119270. [PMID: 33640402 DOI: 10.1016/j.lfs.2021.119270] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Revised: 02/06/2021] [Accepted: 02/14/2021] [Indexed: 02/07/2023]
Abstract
Stem cells (SCs) are clonogenic cells that develop into the specialized cells which later responsible for making up various types of tissue in the human body. SCs are not only the appropriate source of information for cell division, molecular and cellular processes, and tissue homeostasis but also one of the major putative biological aids to diagnose and cure various degenerative diseases. This study emphasises on various research outputs that occurred in the past two decades. This will give brief information on classification, differentiation, detection, and various isolation techniques of SCs. Here, the various signalling pathways which includes WNT, Sonic hedgehog, Notch, BMI1 and C-met pathways and how does it effect on the regeneration of various classes of SCs and factors that regulates the potency of the SCs are also been discussed. We also focused on the application of SCs in the area of regenerative medicine along with the cellular markers that are useful as salient diagnostic or curative tools or in both, by the process of reprogramming, which includes diabetes, cancer, cardiovascular disorders and neurological disorders. The biomarkers that are mentioned in various literatures and experiments include PDX1, FOXA2, HNF6, and NKX6-1 (for diabetes); CD33, CD24, CD133 (for cancer); c-Kit, SCA-1, Wilm's tumor 1 (for cardiovascular disorders); and OCT4, SOX2, c-MYC, EN1, DAT and VMAT2 (for neurological disorders). In this review, we come to know the advancements and scopes of potential SC-based therapies, its diverse applications in clinical fields that can be helpful in the near future.
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Affiliation(s)
- Arnab Sarkar
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Sanjukta Saha
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Abhik Paul
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Avik Maji
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Puspita Roy
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India
| | - Tapan Kumar Maity
- Department of Pharmaceutical Technology, Jadavpur University, West Bengal, Kolkata 700032, India.
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Abstract
Human pluripotent stem cells (hPSCs), either embryonic or induced, offer a plentiful platform for derivation of multiple cell types. Pericytes, generated from hPSCs, are multipotent precursors with vasculogenic features that exhibit high proliferation capability in long-term cultures. Administration of hPSC-pericytes into ischemic murine hind limb is associated with therapeutic angiogenesis and attenuation of muscle wasting. Here, we describe the protocol for derivation of large numbers of pericytes from spontaneously differentiating hPSC-embryoid bodies.
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Oyane A, Araki H, Nakamura M, Aiki Y, Ito Y. Storable bFGF-Releasing Membrane Allowing Continuous Human iPSC Culture. MATERIALS (BASEL, SWITZERLAND) 2021; 14:651. [PMID: 33572553 PMCID: PMC7866866 DOI: 10.3390/ma14030651] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Revised: 01/15/2021] [Accepted: 01/26/2021] [Indexed: 12/26/2022]
Abstract
Basic fibroblast growth factor (bFGF) is a crucial supplement for culture media of human pluripotent stem cells. However, bFGF is extremely unstable under cell culture conditions, which makes frequent (generally every day) medium refreshment requisite. We recently developed a water-floatable, bFGF-releasing membrane via a simple bFGF adsorption process following oxygen plasma treatment by utilizing a polyethylene nonwoven fabric as an adsorbent. This membrane allowed sustained release of bFGF while floating on medium, thereby keeping the bFGF concentration in the medium sufficient for maintaining human-induced pluripotent stem cells (iPSCs) in a proliferative and pluripotent state for as long as 3 days. In this study, lyophilization was applied to the membrane to stabilize bFGF. The sustained bFGF-releasing function of the membrane was kept unchanged even after lyophilization and subsequent cryopreservation at -30 °C for 3 months. The cryopreserved membrane supported proliferation and colony formation of human iPSCs while retaining their viability and pluripotency in a medium-change-free continuous culture for 3 days. The present bFGF-releasing membrane is ready-to-use, storable for at least 3 months, and obviates daily medium refreshment. Therefore, it is a new and more practical bFGF supplement for culture media of human stem cells.
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Affiliation(s)
- Ayako Oyane
- Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan; (H.A.); (M.N.)
| | - Hiroko Araki
- Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan; (H.A.); (M.N.)
| | - Maki Nakamura
- Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan; (H.A.); (M.N.)
| | - Yasuhiko Aiki
- Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan; (Y.A.); (Y.I.)
| | - Yuzuru Ito
- Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba 305-8565, Ibaraki, Japan; (Y.A.); (Y.I.)
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