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Kumar P, Sharma J, Kumar R, Najser J, Frantik J, Sunnam N, Sindhu A, Praveenkumar S. Genetic and bioactive functionalization of bioinks for 3D bioprinting. Bioprocess Biosyst Eng 2025:10.1007/s00449-025-03180-y. [PMID: 40392297 DOI: 10.1007/s00449-025-03180-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: 01/12/2025] [Accepted: 05/06/2025] [Indexed: 05/22/2025]
Abstract
3D bioprinting is revolutionizing tissue engineering and regenerative medicine by enabling the precise fabrication of biologically functional constructs. At its core, the success of 3D bioprinting hinges on the development of bioinks, hydrogel-based materials that support cellular viability, proliferation, and differentiation. However, conventional bioinks face limitations in mechanical strength, biological activity, and customization. Recent advancements in genetic engineering have addressed these challenges by enhancing the properties of bioinks through genetic modifications. These innovations allow the integration of stimuli-responsive elements, bioactive molecules, and extracellular matrix (ECM) components, significantly improving the mechanical integrity, biocompatibility, and functional adaptability of bioinks. This review explores the state-of-the-art genetic approaches to bioink development, emphasizing microbial engineering, genetic functionalization, and the encapsulation of growth factors. It highlights the transformative potential of genetically modified bioinks in various applications, including bone and cartilage regeneration, cardiac and liver tissue engineering, neural tissue reconstruction, and vascularization. While these advances hold promise for personalized and adaptive therapeutic solutions, challenges in scalability, reproducibility, and integration with multi-material systems persist. By bridging genetics and bioprinting, this interdisciplinary field paves the way for sophisticated constructs and innovative therapies in tissue engineering and regenerative medicine.
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Affiliation(s)
- Pawan Kumar
- Department of Biotechnology, Kurukshetra University, Kurukshetra, 136119, India.
| | - Jitender Sharma
- Department of Biotechnology, Kurukshetra University, Kurukshetra, 136119, India
| | - Ravinder Kumar
- Karnavati University, Gandhinagar, 382422, Gujarat, India.
| | - Jan Najser
- ENET Centre, VSB, Technical University of Ostrava, 70800, Ostrava, Czech Republic
| | - Jaroslav Frantik
- ENET Centre, VSB, Technical University of Ostrava, 70800, Ostrava, Czech Republic
| | - Nagaraju Sunnam
- Department of Mechanical Engineering, MLR Institute of Technology, Hyderabad, Telangana, India
| | - Anil Sindhu
- Department of Biotechnology, Deenbandhu Chhotu Ram University of Science and Technology, Murthal, 131039, India
| | - Seepana Praveenkumar
- Department of Nuclear and Renewable Energy, Ural Federal University Named After the First President of Russia Boris, 19 Mira Street, 620002, Ekaterinburg, Yeltsin, Russia
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Rodrigues FAP, Oliveira CS, Sá SC, Tavaria FK, Lee SJ, Oliveira AL, Costa JB. Molecules in Motion: Unravelling the Dynamics of Vascularization Control in Tissue Engineering. Macromol Biosci 2024; 24:e2400139. [PMID: 39422632 DOI: 10.1002/mabi.202400139] [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: 03/25/2024] [Revised: 08/14/2024] [Indexed: 10/19/2024]
Abstract
Significant progress has been made in tissue engineering (TE), aiming at providing personalized solutions and overcoming the current limitations of traditional tissue and organ transplantation. 3D bioprinting has emerged as a transformative technology in the field, able to mimic key properties of the natural architecture of the native tissues. However, most successes in the area are still limited to avascular or thin tissues due to the difficulties in controlling the vascularization of the engineered tissues. To address this issue, several molecules, biomaterials, and cells with pro- and anti-angiogenic potential have been intensively investigated. Furthermore, different bioreactors capable to provide a dynamic environment for in vitro vascularization control have been also explored. The present review summarizes the main molecules and TE strategies used to promote and inhibit vascularization in TE, as well as the techniques used to deliver them. Additionally, it also discusses the current challenges in 3D bioprinting and in tissue maturation to control in vitro/in vivo vascularization. Currently, this field of investigation is of utmost importance and may open doors for the design and development of more precise and controlled vascularization strategies in TE.
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Affiliation(s)
- Francisco A P Rodrigues
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
| | - Cláudia S Oliveira
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
| | - Simone C Sá
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
| | - Freni K Tavaria
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
| | - Sang Jin Lee
- Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA
| | - Ana L Oliveira
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
| | - João B Costa
- CBQF-Centro de Biotecnologia e Química Fina-Laboratório Associado, Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, 4169-005, Portugal
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Wrublewsky S, Schultz J, Ammo T, Bickelmann C, Metzger W, Später T, Pohlemann T, Menger MD, Laschke MW. Biofabrication of prevascularized spheroids for bone tissue engineering by fusion of microvascular fragments with osteoblasts. Front Bioeng Biotechnol 2024; 12:1436519. [PMID: 39318668 PMCID: PMC11419975 DOI: 10.3389/fbioe.2024.1436519] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2024] [Accepted: 08/29/2024] [Indexed: 09/26/2024] Open
Abstract
Introduction Spheroids are promising building blocks for scaffold-free bone tissue engineering. Their rapid vascularization is of major importance to guarantee their survival after transplantation. To achieve this, we herein introduce the biofabrication of prevascularized spheroids by fusion of adipose tissue-derived microvascular fragments (MVF) with osteoblasts (OB). Methods For this purpose, 200 MVF from donor mice and 5,000, 10,000 or 20,000 murine OB (MC3T3-E1) were co-cultured in a liquid overlay system for 3 days to generate OB + MVF spheroids. OB mono-culture spheroids served as controls. Results and discussion During the generation process, the diameters of all spheroids progressively decreased, resulting in compact, viable spheroids of homogeneous sizes. MVF promoted the maturation of spheroids containing 5,000 OB, as shown by an accelerated decline of cell proliferation due to contact inhibition. Moreover, MVF most effectively reassembled into new microvascular networks within these small spheroids when compared to the other spheroid types, indicating the most beneficial MVF to OB ratio. Accordingly, these spheroids also showed a high angiogenic sprouting activity in vitro. In contrast to OB spheroids, they further rapidly vascularized in vivo after transplantation into dorsal skinfold chambers. This was caused by the interconnection of incorporated MVF with surrounding blood vessels. These findings indicate that OB + MVF spheroids may be suitable for bone tissue engineering, which should be next tested in appropriate in vivo bone defect models.
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Affiliation(s)
- Selina Wrublewsky
- Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany
| | - Jessica Schultz
- Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany
- Department of Trauma, Hand, and Reconstructive Surgery, Saarland University, Homburg, Germany
| | - Tekoshin Ammo
- Department of Trauma, Hand, and Reconstructive Surgery, Saarland University, Homburg, Germany
| | - Caroline Bickelmann
- Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany
| | - Wolfgang Metzger
- Department of Trauma, Hand, and Reconstructive Surgery, Saarland University, Homburg, Germany
| | - Thomas Später
- Department of Molecular, Cell and Systems Biology, University of California, Riverside, Riverside, CA, United States
| | - Tim Pohlemann
- Department of Trauma, Hand, and Reconstructive Surgery, Saarland University, Homburg, Germany
| | - Michael D. Menger
- Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany
| | - Matthias W. Laschke
- Institute for Clinical and Experimental Surgery, Saarland University, Homburg, Germany
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Harding J, Vintersten-Nagy K, Yang H, Tang JK, Shutova M, Jong ED, Lee JH, Massumi M, Oussenko T, Izadifar Z, Zhang P, Rogers IM, Wheeler MB, Lye SJ, Sung HK, Li C, Izadifar M, Nagy A. Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts. Nat Biomed Eng 2024; 8:427-442. [PMID: 37996616 PMCID: PMC11087263 DOI: 10.1038/s41551-023-01133-y] [Citation(s) in RCA: 20] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 09/30/2023] [Indexed: 11/25/2023]
Abstract
The immunogenicity of transplanted allogeneic cells and tissues is a major hurdle to the advancement of cell therapies. Here we show that the overexpression of eight immunomodulatory transgenes (Pdl1, Cd200, Cd47, H2-M3, Fasl, Serpinb9, Ccl21 and Mfge8) in mouse embryonic stem cells (mESCs) is sufficient to immunologically 'cloak' the cells as well as tissues derived from them, allowing their survival for months in outbred and allogeneic inbred recipients. Overexpression of the human orthologues of these genes in human ESCs abolished the activation of allogeneic human peripheral blood mononuclear cells and their inflammatory responses. Moreover, by using the previously reported FailSafe transgene system, which transcriptionally links a gene essential for cell division with an inducible and cell-proliferation-dependent kill switch, we generated cloaked tissues from mESCs that served as immune-privileged subcutaneous sites that protected uncloaked allogeneic and xenogeneic cells from rejection in immune-competent hosts. The combination of cloaking and FailSafe technologies may allow for the generation of safe and allogeneically accepted cell lines and off-the-shelf cell products.
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Affiliation(s)
- Jeffrey Harding
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Kristina Vintersten-Nagy
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Huijuan Yang
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Jean Kit Tang
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Maria Shutova
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Eric D Jong
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada
| | - Ju Hee Lee
- Translational Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - Mohammad Massumi
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Tatiana Oussenko
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Zohreh Izadifar
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Puzheng Zhang
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Ian M Rogers
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
- Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada
| | - Michael B Wheeler
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
- Toronto General Hospital Research Institute, Toronto, Ontario, Canada
| | - Stephen J Lye
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
- Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada
| | - Hoon-Ki Sung
- Translational Medicine Program, The Hospital for Sick Children, Toronto, Ontario, Canada
- Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
| | - ChengJin Li
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Mohammad Izadifar
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada
| | - Andras Nagy
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada.
- Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
- Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada.
- Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia.
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Bertucci T, Kakarla S, Winkelman MA, Lane K, Stevens K, Lotz S, Grath A, James D, Temple S, Dai G. Direct differentiation of human pluripotent stem cells into vascular network along with supporting mural cells. APL Bioeng 2023; 7:036107. [PMID: 37564277 PMCID: PMC10411996 DOI: 10.1063/5.0155207] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Accepted: 07/11/2023] [Indexed: 08/12/2023] Open
Abstract
During embryonic development, endothelial cells (ECs) undergo vasculogenesis to form a primitive plexus and assemble into networks comprised of mural cell-stabilized vessels with molecularly distinct artery and vein signatures. This organized vasculature is established prior to the initiation of blood flow and depends on a sequence of complex signaling events elucidated primarily in animal models, but less studied and understood in humans. Here, we have developed a simple vascular differentiation protocol for human pluripotent stem cells that generates ECs, pericytes, and smooth muscle cells simultaneously. When this protocol is applied in a 3D hydrogel, we demonstrate that it recapitulates the dynamic processes of early human vessel formation, including acquisition of distinct arterial and venous fates, resulting in a vasculogenesis angiogenesis model plexus (VAMP). The VAMP captures the major stages of vasculogenesis, angiogenesis, and vascular network formation and is a simple, rapid, scalable model system for studying early human vascular development in vitro.
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Affiliation(s)
| | - Shravani Kakarla
- Northeastern University, Department of Bioengineering, Boston, Massachusetts 02115, USA
| | - Max A. Winkelman
- Northeastern University, Department of Bioengineering, Boston, Massachusetts 02115, USA
| | - Keith Lane
- Neural Stem Cell Institute, Rensselaer, New York 12144, USA
| | | | - Steven Lotz
- Neural Stem Cell Institute, Rensselaer, New York 12144, USA
| | - Alexander Grath
- Northeastern University, Department of Bioengineering, Boston, Massachusetts 02115, USA
| | - Daylon James
- Weill Cornell Medicine, New York, New York 10065, USA
| | - Sally Temple
- Neural Stem Cell Institute, Rensselaer, New York 12144, USA
| | - Guohao Dai
- Northeastern University, Department of Bioengineering, Boston, Massachusetts 02115, USA
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Nguyen V, Gao C, Hochman ML, Kravitz J, Chen EH, Friedman HI, Wenceslau CF, Chen D, Wang Y, Nelson JS, Jegga AG, Tan W. Supporting materials: Endothelial cells differentiated from patient dermal fibroblast-derived induced pluripotent stem cells resemble vascular malformations of Port Wine Birthmark. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.02.547408. [PMID: 37662218 PMCID: PMC10473620 DOI: 10.1101/2023.07.02.547408] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/05/2023]
Abstract
Background Port wine birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models for PWB studies is currently an unmet need. Objective Our study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and iPSC-derived ECs that preserve disease-related phenotypes. Methods PWB iPSCs were generated by reprogramming lesional dermal fibroblasts and differentiated into ECs. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized by capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo . Results Human PWB and control iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the "Yamanaka factors" (Oct3/4, Sox2, Klf4, c-Myc) into them; the iPSCs were successfully differentiated into ECs. These iPSCs and their derived ECs were validated by expression of a series of stem cell and EC biomarkers, respectively. PWB iPSC-derived ECs showed impaired CLS in vitro with larger perimeters and thicker branches as compared to control iPSC-derived ECs. In the plug-in assay, perfused human vasculature formed by PWB iPSC- derived ECs showed bigger perimeters and greater densities than those formed by control iPSC- derived ECs in severe combined immune deficient (SCID) mice. The transcriptome analysis showed that dysregulated pathways of stem cell differentiation, Hippo, Wnt, and focal adhesion persisted through differentiation of PWB iPSCs to ECs. Functional enrichment analysis showed that Hippo and Wnt pathway-related PWB DEGs are enriched for vasculature development, tube morphology, endothelium development, and EC differentiation. Further, members of the zinc finger (ZNF) gene family were overrepresented among the DEGs in PWB iPSCs. ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinoic acid receptor signaling. Furthermore, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were dysregulated in PWB ECs as readouts of impaired differentiation. Conclusions PWB iPSC-derived ECs render a novel and clinically-relevant disease model by retaining pathological phenotypes. Our data demonstrate multiple pathways, such as Hippo and Wnt, NF-kappa B, TNF, MAPK, and cholesterol metabolism, are dysregulated, which may contribute to the development of differentiation-defective ECs in PWB. Bulleted statements What is already known about this topic?: Port Wine Birthmark (PWB) is a congenital vascular malformation with an incidence rate of 0.1 - 0.3 % per live births.PWB results from developmental defects in the dermal vasculature; PWB endothelial cells (ECs) have differentiational impairments.Pulse dye laser (PDL) is currently the preferred treatment for PWB; unfortunately, the efficacy of PDL treatment of PWB has not improved over the past three decades.What does this study add?: Induced pluripotent stem cells (iPSCs) were generated from PWB skin fibroblasts and differentiated into ECs.PWB ECs recapitulated their pathological phenotypes such as forming enlarged blood vessels in vitro and in vivo.Hippo and Wnt pathways were dysregulated in PWB iPSCs and ECs.Zinc-finger family genes were overrepresented among the differentially expressed genes in PWB iPSCs.Dysregulated NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were enriched in PWB ECs.What is the translational message?: Targeting Hippo and Wnt pathways and Zinc-finger family genes could restore the physiological differentiation of ECs.Targeting NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways could mitigate the pathological progression of PWB.These mechanisms may lead to the development of paradigm-shifting therapeutic interventions for PWB.
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Affiliation(s)
- Vi Nguyen
- Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29209, USA
| | - Chao Gao
- Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29209, USA
| | - Marcelo L Hochman
- The Facial Surgery Center and the Hemangioma & Malformation Treatment Center, Charleston, South Carolina 29425, USA
- Department of Otolaryngology - Head and Neck Surgery, Medical University of South Carolina, Charleston, South Carolina 29425 USA
| | - Jacob Kravitz
- Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29209, USA
| | - Elliott H Chen
- Division of Plastic Surgery, School of Medicine, University of South Carolina, Columbia, South Carolina 29203, USA
- Division of Plastic Surgery, Prisma Health Medical Group, Columbia, South Carolina 29203, USA
| | - Harold I Friedman
- Division of Plastic Surgery, School of Medicine, University of South Carolina, Columbia, South Carolina 29203, USA
- Division of Plastic Surgery, Prisma Health Medical Group, Columbia, South Carolina 29203, USA
| | - Camilla F Wenceslau
- Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29209, USA
- Department of Biomedical Engineering, College of Engineering and Computing, University of South Carolina, Columbia, South Carolina 29208, USA
| | - Dongbao Chen
- Department of Obstetrics and Gynecology, University of California, Irvine, Irvine, California 92617, USA
| | - Yunguan Wang
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA
- Division of Gastroenterology, Cincinnati Children Hospital Medical Center, Cincinnati, Ohio 45229, USA
- Division of Human Genetics, Cincinnati Children Hospital Medical Center, Cincinnati, Ohio 45229, USA
| | - J Stuart Nelson
- Departments of Surgery and Biomedical Engineering, Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, California 92617, USA
| | - Anil G. Jegga
- Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45229, USA
- Division of Biomedical Informatics, Cincinnati Children Hospital Medical Center, Cincinnati, Ohio 45229, USA
| | - Wenbin Tan
- Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina 29209, USA
- Department of Biomedical Engineering, College of Engineering and Computing, University of South Carolina, Columbia, South Carolina 29208, USA
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Noh KM, Park SJ, Moon SH, Jung SY. Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells. Front Cardiovasc Med 2023; 10:1169331. [PMID: 37435057 PMCID: PMC10330705 DOI: 10.3389/fcvm.2023.1169331] [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: 02/19/2023] [Accepted: 05/23/2023] [Indexed: 07/13/2023] Open
Abstract
The generation of endothelial cells (ECs) from human pluripotent stem cells (PSCs) has been a promising approach for treating cardiovascular diseases for several years. Human PSCs, particularly induced pluripotent stem cells (iPSCs), are an attractive source of ECs for cell therapy. Although there is a diversity of methods for endothelial cell differentiation using biochemical factors, such as small molecules and cytokines, the efficiency of EC production varies depending on the type and dose of biochemical factors. Moreover, the protocols in which most EC differentiation studies have been performed were in very unphysiological conditions that do not reflect the microenvironment of native tissue. The microenvironment surrounding stem cells exerts variable biochemical and biomechanical stimuli that can affect stem cell differentiation and behavior. The stiffness and components of the extracellular microenvironment are critical inducers of stem cell behavior and fate specification by sensing the extracellular matrix (ECM) cues, adjusting the cytoskeleton tension, and delivering external signals to the nucleus. Differentiation of stem cells into ECs using a cocktail of biochemical factors has been performed for decades. However, the effects of mechanical stimuli on endothelial cell differentiation remain poorly understood. This review provides an overview of the methods used to differentiate ECs from stem cells by chemical and mechanical stimuli. We also propose the possibility of a novel EC differentiation strategy using a synthetic and natural extracellular matrix.
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Affiliation(s)
- Kyung Mu Noh
- Stem Cell Research Institute, T&R Biofab Co. Ltd., Seongnam-si, Republic of Korea
| | - Soon-Jung Park
- Stem Cell Research Institute, T&R Biofab Co. Ltd., Seongnam-si, Republic of Korea
| | - Sung-Hwan Moon
- Department of Animal Science and Technology, College of Biotechnology and Natural Resources, Chung-Ang University, Anseong-si, Republic of Korea
| | - Seok Yun Jung
- Stem Cell Research Institute, T&R Biofab Co. Ltd., Seongnam-si, Republic of Korea
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Ho WJ, Kobayashi M, Murata K, Hashimoto Y, Izumi K, Kimura T, Kanemitsu H, Yamazaki K, Ikeda T, Minatoya K, Kishida A, Masumoto H. A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture. Sci Rep 2022; 12:22294. [PMID: 36566330 PMCID: PMC9789980 DOI: 10.1038/s41598-022-26792-w] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 12/20/2022] [Indexed: 12/25/2022] Open
Abstract
Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.
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Affiliation(s)
- Wen-Jin Ho
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan
| | - Mako Kobayashi
- grid.265073.50000 0001 1014 9130Department of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan ,grid.69566.3a0000 0001 2248 6943Present Address: Department of Materials Processing, Graduate School of Engineering, Tohoku University, Sendai, Japan
| | - Kozue Murata
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan ,grid.508743.dClinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan ,grid.411217.00000 0004 0531 2775Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, Kyoto, Japan
| | - Yoshihide Hashimoto
- grid.265073.50000 0001 1014 9130Department of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
| | | | - Tsuyoshi Kimura
- grid.265073.50000 0001 1014 9130Department of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
| | - Hideo Kanemitsu
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan ,grid.415392.80000 0004 0378 7849Present Address: Department of Cardiovascular Surgery, Kitano Hospital, Osaka, Japan
| | - Kazuhiro Yamazaki
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan
| | - Tadashi Ikeda
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan
| | - Kenji Minatoya
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan
| | - Akio Kishida
- grid.265073.50000 0001 1014 9130Department of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan
| | - Hidetoshi Masumoto
- grid.258799.80000 0004 0372 2033Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto, 606-8507 Japan ,grid.508743.dClinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
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9
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Vargas-Valderrama A, Ponsen AC, Le Gall M, Clay D, Jacques S, Manoliu T, Rouffiac V, Ser-le-Roux K, Quivoron C, Louache F, Uzan G, Mitjavila-Garcia MT, Oberlin E, Guenou H. Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor. Stem Cell Res Ther 2022; 13:254. [PMID: 35715824 PMCID: PMC9205076 DOI: 10.1186/s13287-022-02925-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Accepted: 05/29/2022] [Indexed: 11/10/2022] Open
Abstract
Background hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages.
Methods Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations.
Results A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors.
Conclusion The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02925-w.
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Affiliation(s)
| | - Anne-Charlotte Ponsen
- INSERM UMRS-MD 1197, Hôpital Paul Brousse, Université Paris-Saclay, 94807, Villejuif, France
| | - Morgane Le Gall
- Plateforme Protéomique 3P5-Proteom'IC, Institut Cochin, INSERM U1016, CNRS UMR8104, Université de Paris, 75014, Paris, France
| | - Denis Clay
- INSERM UMS-44, Hôpital Paul Brousse, Université Paris Sud-Université Paris-Saclay, 94807, Villejuif, France
| | - Sébastien Jacques
- Plateforme de Génomique- GENOM'IC, Institut Cochin, INSERM U1016, CNRS UMR8104, Université de Paris, 75014, Paris, France
| | - Tudor Manoliu
- Plate-forme Imagerie et Cytométrie, UMS AMMICa, Gustave Roussy, Université Paris-Saclay, 94805, Villejuif, France
| | - Valérie Rouffiac
- Plate-forme Imagerie et Cytométrie, UMS AMMICa, Gustave Roussy, Université Paris-Saclay, 94805, Villejuif, France
| | - Karine Ser-le-Roux
- INSERM, UMS AMMICa, Plate-forme d'Evaluation Préclinique, Gustave Roussy, 94807, Villejuif, France
| | - Cyril Quivoron
- Laboratoire d'Hématologie Translationnelle, Gustave Roussy, 94805, Villejuif, France
| | - Fawzia Louache
- INSERM UMRS-MD 1197, Hôpital Paul Brousse, Université Paris-Saclay, 94807, Villejuif, France
| | - Georges Uzan
- INSERM UMRS-MD 1197, Hôpital Paul Brousse, Université Paris-Saclay, 94807, Villejuif, France
| | | | - Estelle Oberlin
- INSERM UMRS-MD 1197, Hôpital Paul Brousse, Université Paris-Saclay, 94807, Villejuif, France
| | - Hind Guenou
- INSERM UMRS-MD 1197, Hôpital Paul Brousse, Université Paris-Saclay, 94807, Villejuif, France. .,Université d'Evry-Val-d'Essonne, Université Paris-Saclay, 91000, Evry, France.
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10
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Hamad S, Derichsweiler D, Gaspar JA, Brockmeier K, Hescheler J, Sachinidis A, Pfannkuche KP. High-efficient serum-free differentiation of endothelial cells from human iPS cells. Stem Cell Res Ther 2022; 13:251. [PMID: 35690874 PMCID: PMC9188069 DOI: 10.1186/s13287-022-02924-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Accepted: 05/29/2022] [Indexed: 11/10/2022] Open
Abstract
Introduction Endothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs’ physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications.
Method Human induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h.
Result This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4).
Conclusion The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.
Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02924-x.
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Affiliation(s)
- Sarkawt Hamad
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany.,Biology Department, Faculty of Science, Soran University, Kurdistan Region, Soran, Iraq
| | - Daniel Derichsweiler
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany
| | - John Antonydas Gaspar
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany
| | - Konrad Brockmeier
- Department of Pediatric Cardiology, University Hospital of Cologne, Cologne, Germany
| | - Jürgen Hescheler
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany
| | - Agapios Sachinidis
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany.,Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany
| | - Kurt Paul Pfannkuche
- Medical Faculty, Center for Physiology and Pathophysiology, Institute for Neurophysiology, University of Cologne, Robert Koch Str. 39, 50931, Cologne, Germany. .,Department of Pediatric Cardiology, University Hospital of Cologne, Cologne, Germany. .,Marga-and-Walter-Boll Laboratory for Cardiac Tissue Engineering, University of Cologne, Cologne, Germany. .,Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
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11
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Rogozinski N, Yanez A, Bhoi R, Lee MY, Yang H. Current methods for fabricating 3D cardiac engineered constructs. iScience 2022; 25:104330. [PMID: 35602954 PMCID: PMC9118671 DOI: 10.1016/j.isci.2022.104330] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
3D cardiac engineered constructs have yielded not only the next generation of cardiac regenerative medicine but also have allowed for more accurate modeling of both healthy and diseased cardiac tissues. This is critical as current cardiac treatments are rudimentary and often default to eventual heart transplants. This review serves to highlight the various cell types found in cardiac tissues and how they correspond with current advanced fabrication methods for creating cardiac engineered constructs capable of shedding light on various pathologies and providing the therapeutic potential for damaged myocardium. In addition, insight is given toward the future direction of the field with an emphasis on the creation of specialized and personalized constructs that model the region-specific microtopography and function of native cardiac tissues.
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Affiliation(s)
- Nicholas Rogozinski
- Department of Biomedical Engineering, University of North Texas, 3940 N. Elm Street K240B, Denton, TX 76207-7102, USA
| | - Apuleyo Yanez
- Department of Biomedical Engineering, University of North Texas, 3940 N. Elm Street K240B, Denton, TX 76207-7102, USA
| | - Rahulkumar Bhoi
- Department of Biomedical Engineering, University of North Texas, 3940 N. Elm Street K240B, Denton, TX 76207-7102, USA
| | - Moo-Yeal Lee
- Department of Biomedical Engineering, University of North Texas, 3940 N. Elm Street K240B, Denton, TX 76207-7102, USA
| | - Huaxiao Yang
- Department of Biomedical Engineering, University of North Texas, 3940 N. Elm Street K240B, Denton, TX 76207-7102, USA
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12
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Domingues A, Rossi E, Bujko K, Detriche G, Richez U, Blandinieres A, Kucia M, Ratajczak J, Smadja DM, Ratajczak MZ. Human CD34 + very small embryonic-like stem cells can give rise to endothelial colony-forming cells with a multistep differentiation strategy using UM171 and nicotinamide acid. Leukemia 2022; 36:1440-1443. [PMID: 35169243 PMCID: PMC9061289 DOI: 10.1038/s41375-022-01517-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 01/17/2022] [Accepted: 01/28/2022] [Indexed: 01/25/2023]
Affiliation(s)
- Alison Domingues
- Stem Cell Biology Program, University of Louisville, Louisville, KY, 40245, USA
| | - Elisa Rossi
- Université de Paris, INSERM, Innovative Therapies in Haemostasis, F-75006, Paris, France
| | - Kamila Bujko
- Laboratory of Regenerative Medicine, Medical University of Warsaw, Warsaw, Poland
| | - Grégoire Detriche
- Université de Paris, INSERM, Innovative Therapies in Haemostasis, F-75006, Paris, France
- Vascular Medicine Department and Biosurgical Research Lab (Carpentier Foundation), AP-HP, Hôpital Européen Georges Pompidou, F-75015, Paris, France
| | - Ulysse Richez
- Vascular Medicine Department and Biosurgical Research Lab (Carpentier Foundation), AP-HP, Hôpital Européen Georges Pompidou, F-75015, Paris, France
| | - Adeline Blandinieres
- Université de Paris, INSERM, Innovative Therapies in Haemostasis, F-75006, Paris, France
- Hematology Department and Biosurgical Research Lab (Carpentier Foundation), AP-HP, Hôpital Européen Georges Pompidou, F-75015, Paris, France
| | - Magdalena Kucia
- Stem Cell Biology Program, University of Louisville, Louisville, KY, 40245, USA
- Laboratory of Regenerative Medicine, Medical University of Warsaw, Warsaw, Poland
| | - Janina Ratajczak
- Stem Cell Biology Program, University of Louisville, Louisville, KY, 40245, USA
| | - David M Smadja
- Université de Paris, INSERM, Innovative Therapies in Haemostasis, F-75006, Paris, France.
- Vascular Medicine Department and Biosurgical Research Lab (Carpentier Foundation), AP-HP, Hôpital Européen Georges Pompidou, F-75015, Paris, France.
| | - Mariusz Z Ratajczak
- Stem Cell Biology Program, University of Louisville, Louisville, KY, 40245, USA.
- Laboratory of Regenerative Medicine, Medical University of Warsaw, Warsaw, Poland.
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13
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Tracy EP, Stielberg V, Rowe G, Benson D, Nunes SS, Hoying JB, Murfee WL, LeBlanc AJ. State of the field: cellular and exosomal therapeutic approaches in vascular regeneration. Am J Physiol Heart Circ Physiol 2022; 322:H647-H680. [PMID: 35179976 PMCID: PMC8957327 DOI: 10.1152/ajpheart.00674.2021] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/14/2021] [Revised: 02/07/2022] [Accepted: 02/09/2022] [Indexed: 01/19/2023]
Abstract
Pathologies of the vasculature including the microvasculature are often complex in nature, leading to loss of physiological homeostatic regulation of patency and adequate perfusion to match tissue metabolic demands. Microvascular dysfunction is a key underlying element in the majority of pathologies of failing organs and tissues. Contributing pathological factors to this dysfunction include oxidative stress, mitochondrial dysfunction, endoplasmic reticular (ER) stress, endothelial dysfunction, loss of angiogenic potential and vascular density, and greater senescence and apoptosis. In many clinical settings, current pharmacologic strategies use a single or narrow targeted approach to address symptoms of pathology rather than a comprehensive and multifaceted approach to address their root cause. To address this, efforts have been heavily focused on cellular therapies and cell-free therapies (e.g., exosomes) that can tackle the multifaceted etiology of vascular and microvascular dysfunction. In this review, we discuss 1) the state of the field in terms of common therapeutic cell population isolation techniques, their unique characteristics, and their advantages and disadvantages, 2) common molecular mechanisms of cell therapies to restore vascularization and/or vascular function, 3) arguments for and against allogeneic versus autologous applications of cell therapies, 4) emerging strategies to optimize and enhance cell therapies through priming and preconditioning, and, finally, 5) emerging strategies to bolster therapeutic effect. Relevant and recent clinical and animal studies using cellular therapies to restore vascular function or pathologic tissue health by way of improved vascularization are highlighted throughout these sections.
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Affiliation(s)
- Evan Paul Tracy
- Cardiovascular Innovation Institute and the Department of Physiology, University of Louisville, Louisville, Kentucky
| | - Virginia Stielberg
- Cardiovascular Innovation Institute and the Department of Physiology, University of Louisville, Louisville, Kentucky
| | - Gabrielle Rowe
- Cardiovascular Innovation Institute and the Department of Physiology, University of Louisville, Louisville, Kentucky
| | - Daniel Benson
- Cardiovascular Innovation Institute and the Department of Physiology, University of Louisville, Louisville, Kentucky
- Department of Bioengineering, University of Louisville, Louisville, Kentucky
| | - Sara S Nunes
- Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
- Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
- Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, Toronto, Ontario, Canada
| | - James B Hoying
- Advanced Solutions Life Sciences, Manchester, New Hampshire
| | - Walter Lee Murfee
- J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, Florida
| | - Amanda Jo LeBlanc
- Cardiovascular Innovation Institute and the Department of Physiology, University of Louisville, Louisville, Kentucky
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14
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Yoshimi R, Nakajima H. Current State and Issues of Regenerative Medicine for Rheumatic Diseases. Front Med (Lausanne) 2022; 9:813952. [PMID: 35155499 PMCID: PMC8831787 DOI: 10.3389/fmed.2022.813952] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2021] [Accepted: 01/05/2022] [Indexed: 12/15/2022] Open
Abstract
The prognosis of rheumatic diseases is generally better than that of malignant diseases. However, some cases with poor prognoses resist conventional therapies and cause irreversible functional and organ damage. In recent years, there has been much research on regenerative medicine, which uses stem cells to restore the function of missing or dysfunctional tissues and organs. The development of regenerative medicine is also being attempted in rheumatic diseases. In diseases such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), and rheumatoid arthritis, hematopoietic stem cell transplantation has been attempted to correct and reconstruct abnormalities in the immune system. Mesenchymal stem cells (MSCs) have also been tried for the treatment of refractory skin ulcers in SSc using the ability of MSCs to differentiate into vascular endothelial cells and for the treatment of systemic lupus erythematosus SLE using the immunosuppressive effect of MSCs. CD34-positive endothelial progenitor cells (EPCs), which are found in the mononuclear cell fraction of bone marrow and peripheral blood, can differentiate into vascular endothelial cells at the site of ischemia. Therefore, EPCs have been used in research on vascular regeneration therapy for patients with severe lower limb ischemia caused by rheumatic diseases such as SSc. Since the first report of induced pluripotent stem cells (iPSCs) in 2007, research on regenerative medicine using iPSCs has been actively conducted, and their application to rheumatic diseases is expected. However, there are many safety issues and bioethical issues involved in regenerative medicine research, and it is essential to resolve these issues for practical application and spread of regenerative medicine in the future. The environment surrounding regenerative medicine research is changing drastically, and the required expertise is becoming higher. This paper outlines the current status and challenges of regenerative medicine in rheumatic diseases.
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15
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Pavlova M, McGarvey SS, Bilousova G, Kogut I. A High-Efficiency Method for the Production of Endothelial Cells from Human Induced Pluripotent Stem Cells. Methods Mol Biol 2022; 2549:169-186. [PMID: 33755906 PMCID: PMC8460679 DOI: 10.1007/7651_2021_377] [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: 01/03/2023]
Abstract
Endothelial cells (ECs) are important components of the circulatory system. These cells can be used for in vitro modeling of cardiovascular diseases and in regenerative medicine to promote vascularization of engineered tissue constructs. However, low proliferative capacity and patient-to-patient variability limit the use of primary ECs in the clinic and disease modeling. ECs differentiated from human induced pluripotent stem cells (iPSCs) can serve as a viable alternative to primary ECs for these applications. This is because human iPSCs can proliferate indefinitely and have the potential to differentiate into a variety of somatic cell lines, providing a renewable source of patient-specific cells. Here, we present an optimized, highly reproducible method for the differentiation of human iPSCs toward vascular ECs. The protocol relies on the activation of the WNT signaling pathway and the use of growth factors and small molecules. The resulting iPSC-derived ECs can be cultured for multiple passages without losing their functionality and are suitable for both in vitro and in vivo studies.
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Affiliation(s)
- Maryna Pavlova
- Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA
| | - Shennea S. McGarvey
- Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA
| | - Ganna Bilousova
- Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Linda Crnic Institute for Down Syndrome, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA
| | - Igor Kogut
- Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, Colorado, USA,Correspondence: Igor Kogut, Charles C. Gates Center for Regenerative Medicine, University of Colorado School of Medicine, Anschutz Medical Campus, 12700 E. 19 Ave, Research Complex 2, P15-4004, Aurora, CO 80045. Phone: 303-724-6141; Fax: 303-724-3051;
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16
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Moreira A, Müller M, Costa PF, Kohl Y. Advanced In Vitro Lung Models for Drug and Toxicity Screening: The Promising Role of Induced Pluripotent Stem Cells. Adv Biol (Weinh) 2021; 6:e2101139. [PMID: 34962104 DOI: 10.1002/adbi.202101139] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Revised: 11/25/2021] [Indexed: 12/24/2022]
Abstract
The substantial socioeconomic burden of lung diseases, recently highlighted by the disastrous impact of the coronavirus disease 2019 (COVID-19) pandemic, accentuates the need for interventive treatments capable of decelerating disease progression, limiting organ damage, and contributing to a functional tissue recovery. However, this is hampered by the lack of accurate human lung research models, which currently fail to reproduce the human pulmonary architecture and biochemical environment. Induced pluripotent stem cells (iPSCs) and organ-on-chip (OOC) technologies possess suitable characteristics for the generation of physiologically relevant in vitro lung models, allowing for developmental studies, disease modeling, and toxicological screening. Importantly, these platforms represent potential alternatives for animal testing, according to the 3Rs (replace, reduce, refine) principle, and hold promise for the identification and approval of new chemicals under the European REACH (registration, evaluation, authorization and restriction of chemicals) framework. As such, this review aims to summarize recent progress made in human iPSC- and OOC-based in vitro lung models. A general overview of the present applications of in vitro lung models is presented, followed by a summary of currently used protocols to generate different lung cell types from iPSCs. Lastly, recently developed iPSC-based lung models are discussed.
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Affiliation(s)
| | - Michelle Müller
- Department of Bioprocessing and Bioanalytics, Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280, Sulzbach, Germany
| | - Pedro F Costa
- BIOFABICS, Rua Alfredo Allen 455, Porto, 4200-135, Portugal
| | - Yvonne Kohl
- Department of Bioprocessing and Bioanalytics, Fraunhofer Institute for Biomedical Engineering IBMT, Joseph-von-Fraunhofer-Weg 1, 66280, Sulzbach, Germany.,Postgraduate Course for Toxicology and Environmental Toxicology, Medical Faculty, University of Leipzig, Johannisallee 28, 04103, Leipzig, Germany
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17
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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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18
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Rauti R, Shahoha M, Leichtmann-Bardoogo Y, Nasser R, Paz E, Tamir R, Miller V, Babich T, Shaked K, Ehrlich A, Ioannidis K, Nahmias Y, Sharan R, Ashery U, Maoz BM. Effect of SARS-CoV-2 proteins on vascular permeability. eLife 2021; 10:69314. [PMID: 34694226 PMCID: PMC8545399 DOI: 10.7554/elife.69314] [Citation(s) in RCA: 43] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2021] [Accepted: 10/09/2021] [Indexed: 12/13/2022] Open
Abstract
Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein–protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.
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Affiliation(s)
- Rossana Rauti
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel
| | - Meishar Shahoha
- School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.,Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
| | | | - Rami Nasser
- Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel
| | - Eyal Paz
- School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.,Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
| | - Rina Tamir
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel
| | - Victoria Miller
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel
| | - Tal Babich
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel.,School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
| | - Kfir Shaked
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel.,School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
| | - Avner Ehrlich
- Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel
| | | | - Yaakov Nahmias
- Grass Center for Bioengineering, The Hebrew University of Jerusalem, Jerusalem, Israel
| | - Roded Sharan
- Blavatnik School of Computer Science, Tel Aviv University, Tel Aviv, Israel
| | - Uri Ashery
- School of Neurobiology, Biochemistry and Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.,Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.,The Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel
| | - Ben Meir Maoz
- Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, Israel.,Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.,The Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel
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19
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Hsieh HL, Liang CC, Lu CY, Yang JT, Chung CY, Ko YS, Lee TH. Induced pluripotent stem cells can improve thrombolytic effect of low-dose rt-PA after acute carotid thrombosis in rat. Stem Cell Res Ther 2021; 12:549. [PMID: 34674761 PMCID: PMC8532293 DOI: 10.1186/s13287-021-02615-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Accepted: 10/06/2021] [Indexed: 12/17/2022] Open
Abstract
Background Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is the standard treatment for acute ischemic stroke. Standard-dose rt-PA (0.9 mg/kg) is known to achieve good recanalization but carries a high bleeding risk. Lower dose of rt-PA has less bleeding risk but carries a high re-occlusion rate. We investigate if induced pluripotent stem cells (iPSCs) can improve the thrombolytic effect of low-dose rt-PA (0.45 mg/kg). Methods Single irradiation with 6 mW/cm2 light-emitting diode (LED) for 4 h at rat common carotid artery was used as thrombosis model according to our previous report. Endothelin-1 (ET-1), intercellular adhesion molecule-1 (ICAM-1), and interleukin 1 beta (IL-1 beta) were used as the inflammatory markers for artery endothelial injury. Angiopoietin-2 (AP-2), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were examined in artery wall and iPSCs culture. Animal ultrasound was used to evaluate the stenosis degree of common carotid artery before and at 2 h, 24 h, 4 days and 7 days after LED irradiation. Results After LED irradiation alone, there was a persistent occlusion from 2 h to 7 days. Standard-dose rt-PA alone could recanalize the occluded artery from 24 h to 7 days to stenotic degree ≤ 50%. Low-dose rt-PA or 1 × 106 mouse iPSCs alone could not recanalize the occluded arteries from 2 h to 7 days. Combination use of low-dose rt-PA plus 1 × 106 mouse iPSCs caused better recanalization from 24 h to 7 days. ET-1, ICAM-1 and IL-1 beta were strongly expressed after LED irradiation but reduced after iPSCs treatment. AP-2, BDNF and VEGF were rarely induced after LED irradiation but strongly expressed after iPSCs treatment. In vitro study showed iPSCs could express AP-2, BDNF and VEGF. Conclusion The adjuvant use of iPSCs may help improving the thrombolytic effect of low-dose rt-PA by suppressing inflammatory factors and inducing angiogenic trophic factors. Stem cells could be a potential regimen in acute thrombolytic therapy to improve recanalization and reduce complications. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02615-z.
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Affiliation(s)
- Hsi-Lung Hsieh
- Department of Nursing, Division of Basic Medical Sciences, Research Center for Chinese Herbal Medicine, and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan
| | - Ching-Chung Liang
- Female Urology Section, Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Linkou Medical Center, and College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Cheng-You Lu
- Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan
| | - Jen-Tsung Yang
- Department of Neurosurgery, Chia-Yi Chang Gung Memorial Hospital, Chia-Yi, Taiwan, and College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Chiu-Yen Chung
- Department of Neurosurgery, Chia-Yi Chang Gung Memorial Hospital, Chia-Yi, Taiwan, and College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Yu-Shien Ko
- The First Cardiovascular Division, Department of Internal Medicine, Chang Gung Memorial Hospital, Linkou Medical Center, and College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Tsong-Hai Lee
- Stroke Center and Department of Neurology, Chang Gung Memorial Hospital, Linkou Medical Center, and College of Medicine, Chang Gung University, No. 5, Fu-Hsing Street, Kweishan, Taoyuan, 333, Taiwan.
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20
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Helle E, Ampuja M, Dainis A, Antola L, Temmes E, Tolvanen E, Mervaala E, Kivelä R. HiPS-Endothelial Cells Acquire Cardiac Endothelial Phenotype in Co-culture With hiPS-Cardiomyocytes. Front Cell Dev Biol 2021; 9:715093. [PMID: 34422835 PMCID: PMC8378235 DOI: 10.3389/fcell.2021.715093] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2021] [Accepted: 07/14/2021] [Indexed: 01/05/2023] Open
Abstract
Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development.
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Affiliation(s)
- Emmi Helle
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.,New Children's Hospital, Pediatric Research Center, Helsinki University Hospital, Helsinki, Finland
| | - Minna Ampuja
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Alexandra Dainis
- Department of Genetics, Stanford University, Stanford, CA, United States
| | - Laura Antola
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Elina Temmes
- Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki, Finland.,Samaria Health Centre, Espoo, Finland
| | - Erik Tolvanen
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Eero Mervaala
- Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Riikka Kivelä
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.,Wihuri Research Institute, Helsinki, Finland
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21
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Huang Y, Qian JY, Cheng H, Li XM. Effects of shear stress on differentiation of stem cells into endothelial cells. World J Stem Cells 2021; 13:894-913. [PMID: 34367483 PMCID: PMC8316872 DOI: 10.4252/wjsc.v13.i7.894] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 04/20/2021] [Accepted: 06/22/2021] [Indexed: 02/06/2023] Open
Abstract
Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.
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Affiliation(s)
- Yan Huang
- Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Jia-Yi Qian
- Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Hong Cheng
- Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China
| | - Xiao-Ming Li
- Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, Beijing Advanced Innovation Center for Biomedical Engineering, School of Biological Science and Medical Engineering, Beihang University, Beijing 100083, China.
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22
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Kovina MV, Dyuzheva TG, Krasheninnikov ME, Yakovenko SA, Khodarovich YM. Co-growth of Stem Cells With Target Tissue Culture as an Easy and Effective Method of Directed Differentiation. Front Bioeng Biotechnol 2021; 9:591775. [PMID: 34222206 PMCID: PMC8242343 DOI: 10.3389/fbioe.2021.591775] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2020] [Accepted: 02/10/2021] [Indexed: 11/13/2022] Open
Abstract
The long-term co-culture of mouse embryonic stem cells (mESC) with rat endothelial cells (EC) was tested for contact differentiation into the endothelial lineage. Serial passaging of rat ECs mixed with mESC in ratio 10:1 resulted in the emergence of a homogeneous cell population expressing mouse endothelial surface markers CD102, CD29, CD31. Rat endothelial surface marker RECA-1 completely disappeared from the co-cultured population after 2 months of weekly passaging. Co-incubation of mESC with rat ECs without cell-to-cell contact did not result in the conversion of mESC into ECs. After co-cultivation of adult mesenchymal stem cells from human endometrium (eMSC) with pre-hepatocyte-like cells of human hepatocarcinoma Huh7 the resulting co-culture expressed mature liver markers (oval cell antigen and cytokeratin 7), none of which were expressed by any of co-cultivated cultures, thus proving that even an immature (proliferating) pre-hepatocyte-like line can induce hepatic differentiation of stem cells. In conclusion, we have developed conditions where long-term co-proliferation of embryonic or adult SC with fully or partially differentiated cells results in stem cell progeny expressing markers of target tissue. In the case of endothelial differentiation, the template population quickly disappeared from the resulted culture and the pure endothelial population of stem cell progeny emerged. This approach demonstrates the expected fate of stem cells during various in vivo SC-therapies and also might be used as an effective in vitro differentiation method to develop the pure endothelium and, potentially, other tissue types of desirable genetic background.
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Affiliation(s)
- Marina Valentinovna Kovina
- Peoples’ Friendship University of Russia, Moscow, Russia
- AltraVita IVF Clinic, Moscow, Russia
- The University of Texas Health Science Center at Houston, Medical School, Department of Integrative Biology and Pharmacology, Houston, TX, United States
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23
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Gao Y, Pu J. Differentiation and Application of Human Pluripotent Stem Cells Derived Cardiovascular Cells for Treatment of Heart Diseases: Promises and Challenges. Front Cell Dev Biol 2021; 9:658088. [PMID: 34055788 PMCID: PMC8149736 DOI: 10.3389/fcell.2021.658088] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 03/25/2021] [Indexed: 12/15/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) are derived from human embryos (human embryonic stem cells) or reprogrammed from human somatic cells (human induced pluripotent stem cells). They can differentiate into cardiovascular cells, which have great potential as exogenous cell resources for restoring cardiac structure and function in patients with heart disease or heart failure. A variety of protocols have been developed to generate and expand cardiovascular cells derived from hPSCs in vitro. Precisely and spatiotemporally activating or inhibiting various pathways in hPSCs is required to obtain cardiovascular lineages with high differentiation efficiency. In this concise review, we summarize the protocols of differentiating hPSCs into cardiovascular cells, highlight their therapeutic application for treatment of cardiac diseases in large animal models, and discuss the challenges and limitations in the use of cardiac cells generated from hPSCs for a better clinical application of hPSC-based cardiac cell therapy.
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Affiliation(s)
- Yu Gao
- Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Jun Pu
- Department of Cardiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
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24
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Nelson EA, Qiu J, Chavkin NW, Hirschi KK. Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells. J Vis Exp 2021:10.3791/62391. [PMID: 33871448 PMCID: PMC8675434 DOI: 10.3791/62391] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022] Open
Abstract
Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3β inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.
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Affiliation(s)
- Elizabeth A Nelson
- Department of Cell Biology, University of Virginia; Cardiovascular Research Center, University of Virginia
| | - Jingyao Qiu
- Department of Medicine, Yale University School of Medicine; Department of Genetics, Yale University School of Medicine
| | - Nicholas W Chavkin
- Department of Cell Biology, University of Virginia; Cardiovascular Research Center, University of Virginia
| | - Karen K Hirschi
- Department of Cell Biology, University of Virginia; Cardiovascular Research Center, University of Virginia; Department of Medicine, Yale University School of Medicine; Department of Genetics, Yale University School of Medicine; Yale Cardiovascular Research Center, Yale University School of Medicine;
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25
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Induced Pluripotent Stem Cells (iPSCs) in Vascular Research: from Two- to Three-Dimensional Organoids. Stem Cell Rev Rep 2021; 17:1741-1753. [PMID: 33738695 PMCID: PMC7972819 DOI: 10.1007/s12015-021-10149-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/04/2021] [Indexed: 01/19/2023]
Abstract
Stem cell technology has been around for almost 30 years and in that time has grown into an enormous field. The stem cell technique progressed from the first successful isolation of mammalian embryonic stem cells (ESCs) in the 1990s, to the production of human induced-pluripotent stem cells (iPSCs) in the early 2000s, to finally culminate in the differentiation of pluripotent cells into highly specialized cell types, such as neurons, endothelial cells (ECs), cardiomyocytes, fibroblasts, and lung and intestinal cells, in the last decades. In recent times, we have attained a new height in stem cell research whereby we can produce 3D organoids derived from stem cells that more accurately mimic the in vivo environment. This review summarizes the development of stem cell research in the context of vascular research ranging from differentiation techniques of ECs and smooth muscle cells (SMCs) to the generation of vascularized 3D organoids. Furthermore, the different techniques are critically reviewed, and future applications of current 3D models are reported.
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26
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Nalbach L, Roma LP, Schmitt BM, Becker V, Körbel C, Wrublewsky S, Pack M, Später T, Metzger W, Menger MM, Frueh FS, Götz C, Lin H, EM Fox J, MacDonald PE, Menger MD, Laschke MW, Ampofo E. Improvement of islet transplantation by the fusion of islet cells with functional blood vessels. EMBO Mol Med 2021; 13:e12616. [PMID: 33135383 PMCID: PMC7799357 DOI: 10.15252/emmm.202012616] [Citation(s) in RCA: 64] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 09/25/2020] [Accepted: 09/28/2020] [Indexed: 12/12/2022] Open
Abstract
Pancreatic islet transplantation still represents a promising therapeutic strategy for curative treatment of type 1 diabetes mellitus. However, a limited number of organ donors and insufficient vascularization with islet engraftment failure restrict the successful transfer of this approach into clinical practice. To overcome these problems, we herein introduce a novel strategy for the generation of prevascularized islet organoids by the fusion of pancreatic islet cells with functional native microvessels. These insulin-secreting organoids exhibit a significantly higher angiogenic activity compared to freshly isolated islets, cultured islets, and non-prevascularized islet organoids. This is caused by paracrine signaling between the β-cells and the microvessels, mediated by insulin binding to its corresponding receptor on endothelial cells. In vivo, the prevascularized islet organoids are rapidly blood-perfused after transplantation by the interconnection of their autochthonous microvasculature with surrounding blood vessels. As a consequence, a lower number of islet grafts are required to restore normoglycemia in diabetic mice. Thus, prevascularized islet organoids may be used to improve the success rates of clinical islet transplantation.
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Affiliation(s)
- Lisa Nalbach
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Leticia P Roma
- Biophysics DepartmentCenter for Human and Molecular BiologySaarland UniversityHomburg/SaarGermany
| | - Beate M Schmitt
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Vivien Becker
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Christina Körbel
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Selina Wrublewsky
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Mandy Pack
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Thomas Später
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Wolfgang Metzger
- Department of Trauma, Hand and Reconstructive SurgerySaarland UniversityHomburgGermany
| | - Maximilian M Menger
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
- Departement of Trauma and Reconstructive SurgeryEberhar Karls University TuebingenTuebingenGermany
| | - Florian S Frueh
- Division of Plastic Surgery and Hand SurgeryUniversity Hospital ZurichUniversity of ZurichZurichSwitzerland
| | - Claudia Götz
- Medical Biochemistry and Molecular BiologySaarland UniversityHomburgGermany
| | - Haopeng Lin
- Department of PharmacologyAlberta Diabetes InstituteUniversity of AlbertaEdmontonABCanada
| | - Joseline EM Fox
- Department of PharmacologyAlberta Diabetes InstituteUniversity of AlbertaEdmontonABCanada
| | - Patrick E MacDonald
- Department of PharmacologyAlberta Diabetes InstituteUniversity of AlbertaEdmontonABCanada
| | - Michael D Menger
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Matthias W Laschke
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
| | - Emmanuel Ampofo
- Institute for Clinical & Experimental SurgerySaarland UniversityHomburg/SaarGermany
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27
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Ozdogan CY, Kenar H, Davun KE, Yucel D, Doger E, Alagoz S. An in vitro 3D diabetic human skin model from diabetic primary cells. Biomed Mater 2020; 16:015027. [PMID: 33331294 DOI: 10.1088/1748-605x/abc1b1] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Diabetes mellitus, a complex metabolic disorder, leads to many health complications like kidney failure, diabetic heart disease, stroke, and foot ulcers. Treatment approaches of diabetes and identification of the mechanisms underlying diabetic complications of the skin have gained importance due to continued rapid increase in the diabetes incidence. A thick and pre-vascularized in vitro 3D type 2 diabetic human skin model (DHSM) was developed in this study. The methacrylated gelatin (GelMA) hydrogel was produced by photocrosslinking and its pore size (54.85 ± 8.58 μm), compressive modulus (4.53 ± 0.67 kPa) and swelling ratio (17.5 ± 2.2%) were found to be suitable for skin tissue engineering. 8% GelMA hydrogel effectively supported the viability, spreading and proliferation of human dermal fibroblasts. By isolating dermal fibroblasts, human umbilical vein endothelial cells and keratinocytes from type 2 diabetic patients, an in vitro 3D type 2 DHSM, 12 mm in width and 1.86 mm thick, was constructed. The skin model consisted of a continuous basal epidermal layer and a dermal layer with blood capillary-like structures, ideal for evaluating the effects of anti-diabetic drugs and wound healing materials and factors. The functionality of the DHSM was showed by applying a therapeutic hydrogel into its central wound; especially fibroblast migration to the wound site was evident in 9 d. We have demonstrated that DHSM is a biologically relevant model with sensitivity and predictability in evaluating the diabetic wound healing potential of a therapeutic material.
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Affiliation(s)
- Candan Yilmaz Ozdogan
- Experimental and Clinical Research Center, Diabetes and Obesity Research Laboratory, Kocaeli University, Kocaeli, Turkey. Department of Biology, Graduate School of Natural and Applied Sciences, Kocaeli University, Kocaeli, Turkey
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28
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Fukushima H, Yoshioka M, Kawatou M, López-Dávila V, Takeda M, Kanda Y, Sekino Y, Yoshida Y, Yamashita JK. Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells. PLoS One 2020; 15:e0241287. [PMID: 33137106 PMCID: PMC7605685 DOI: 10.1371/journal.pone.0241287] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Accepted: 10/13/2020] [Indexed: 12/28/2022] Open
Abstract
Currently, cardiomyocyte (CM) differentiation methods require a purification step after CM induction to ensure the high purity of the cell population. Here we show an improved human CM differentiation protocol with which high-purity ventricular-type CMs can be obtained and maintained without any CM purification process. We induced and collected a mesodermal cell population (platelet-derived growth factor receptor-α (PDGFRα)-positive cells) that can respond to CM differentiation cues, and then stimulated CM differentiation by means of Wnt inhibition. This method reproducibly generated CMs with purities above 95% in several human pluripotent stem cell lines. Furthermore, these CM populations were maintained in culture at such high purity without any further CM purification step for over 200 days. The majority of these CMs (>95%) exhibited a ventricular-like phenotype with a tendency to structural and electrophysiological maturation, including T-tubule-like structure formation and the ability to respond to QT prolongation drugs. This is a simple and valuable method to stably generate CM populations suitable for cardiac toxicology testing, disease modeling and regenerative medicine.
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Affiliation(s)
- Hiroyuki Fukushima
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Miki Yoshioka
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Masahide Kawatou
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
- Institute for Advancement of Clinical and Translational Science (iACT), Kyoto University Hospital, Kyoto, Japan
| | - Víctor López-Dávila
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Masafumi Takeda
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- Institute for Advancement of Clinical and Translational Science (iACT), Kyoto University Hospital, Kyoto, Japan
| | - Yasunari Kanda
- Division of Pharmacology, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
| | - Yuko Sekino
- Division of Pharmacology, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan
| | - Yoshinori Yoshida
- Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
| | - Jun K. Yamashita
- Department of Cell Growth and Differentiation, Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
- * E-mail:
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29
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Helle E, Ampuja M, Antola L, Kivelä R. Flow-Induced Transcriptomic Remodeling of Endothelial Cells Derived From Human Induced Pluripotent Stem Cells. Front Physiol 2020; 11:591450. [PMID: 33178051 PMCID: PMC7593792 DOI: 10.3389/fphys.2020.591450] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2020] [Accepted: 09/16/2020] [Indexed: 12/31/2022] Open
Abstract
The vascular system is essential for the development and function of all organs and tissues in our body. The molecular signature and phenotype of endothelial cells (EC) are greatly affected by blood flow-induced shear stress, which is a vital component of vascular development and homeostasis. Recent advances in differentiation of ECs from human induced pluripotent stem cells (hiPSC) have enabled development of in vitro experimental models of the vasculature containing cells from healthy individuals or from patients harboring genetic variants or diseases of interest. Here we have used hiPSC-derived ECs and bulk- and single-cell RNA sequencing to study the effect of flow on the transcriptomic landscape of hiPSC-ECs and their heterogeneity. We demonstrate that hiPS-ECs are plastic and they adapt to flow by expressing known flow-induced genes. Single-cell RNA sequencing showed that flow induced a more homogenous and homeostatically more stable EC population compared to static cultures, as genes related to cell polarization, barrier formation and glucose and fatty acid transport were induced. The hiPS-ECs increased both arterial and venous markers when exposed to flow. Interestingly, while in general there was a greater increase in the venous markers, one cluster with more arterial-like hiPS-ECs was detected. Single-cell RNA sequencing revealed that not all hiPS-ECs are similar even after sorting, but exposing them to flow increases their homogeneity. Since hiPS-ECs resemble immature ECs and demonstrate high plasticity in response to flow, they provide an excellent model to study vascular development.
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Affiliation(s)
- Emmi Helle
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- New Children’s Hospital, and Pediatric Research Center Helsinki University Hospital, Helsinki, Finland
| | - Minna Ampuja
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Laura Antola
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
| | - Riikka Kivelä
- Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland
- Wihuri Research Institute, Helsinki, Finland
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Yong U, Lee S, Jung S, Jang J. Interdisciplinary approaches to advanced cardiovascular tissue engineering: ECM-based biomaterials, 3D bioprinting, and its assessment. ACTA ACUST UNITED AC 2020. [DOI: 10.1088/2516-1091/abb211] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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Yi B, Dissanayaka WL, Zhang C. Growth Factors and Small-molecule Compounds in Derivation of Endothelial Lineages from Dental Stem Cells. J Endod 2020; 46:S63-S70. [PMID: 32950197 DOI: 10.1016/j.joen.2020.06.024] [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: 10/23/2022]
Abstract
INTRODUCTION Incorporating fully assembled microvascular networks into bioengineered dental pulp constructs can significantly enhance functional blood flow and tissue survival upon transplantation. Endothelial cells (ECs), cellular building blocks of vascular tissue, play an essential role in the process of prevascularization. However, obtaining sufficient ECs from a suitable source for translational application is challenging. Dental stem cells (DSCs), which exhibit a robust proliferative ability and immunocompatibility because of their autologous origin, could be a promising alternative cell source for the derivation of endothelial lineages. Under specific culture conditions, DSCs differentiate into osteo/odontogenic, adipogenic, chondrogenic, and neurogenic cell lineages. METHODS Recently, a new approach has been developed to directly reprogram cells using chemical cocktails and growth factors. Compared with the traditional reprogramming approach based on the forced expression of exogenous transcription factors, the chemical strategy avoids the risk associated with lentiviral transduction while offering a more viable methodology to drive cell lineage switch. The aim of this review was to unveil the concept of the use of small-molecule compounds and growth factors modulating key signaling pathways to derive ECs from DSCs. RESULTS In addition, our preliminary study showed that stem cells from the apical papilla could be induced into EC-like cells using small-molecule compounds and growth factors. These EC-like cells expressed endothelial specific genes (CD31 and VEGFR2) and proteins (CD31, VEGF receptor 2, and vascular endothelial cadherin) as well as gave rise to vessel-like tubular structures in vitro. CONCLUSIONS Our preliminary results suggest that chemical reprogramming might offer a novel way to generate EC-like cells from dental stem cells.
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Affiliation(s)
- Baicheng Yi
- Restorative Dental Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Special Administrative Region, China
| | - Waruna Lakmal Dissanayaka
- Restorative Dental Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Special Administrative Region, China
| | - Chengfei Zhang
- Restorative Dental Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, Special Administrative Region, China.
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Wei R, Lv J, Li X, Li Y, Xu Q, Jin J, Zhang Y, Liu Z. Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system. J Vet Sci 2020; 21:e9. [PMID: 31940688 PMCID: PMC7000895 DOI: 10.4142/jvs.2020.21.e9] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2019] [Revised: 11/05/2019] [Accepted: 11/21/2019] [Indexed: 12/21/2022] Open
Abstract
Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3β inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.
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Affiliation(s)
- Renyue Wei
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Jiawei Lv
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Xuechun Li
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Yan Li
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Qianqian Xu
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Junxue Jin
- College of Life Science, Northeast Agricultural University, Harbin 150030, China
| | - Yu Zhang
- College of Life Science, Northeast Agricultural University, Harbin 150030, China.
| | - Zhonghua Liu
- College of Life Science, Northeast Agricultural University, Harbin 150030, China.
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Sriram G, K Handral H, Uin Gan S, Islam I, Jalil Rufaihah A, Cao T. Fabrication of vascularized tissue constructs under chemically defined culture conditions. Biofabrication 2020; 12:045015. [DOI: 10.1088/1758-5090/aba0c2] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
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Abstract
Vascularization is a major hurdle in complex tissue and organ engineering. Tissues greater than 200 μm in diameter cannot rely on simple diffusion to obtain nutrients and remove waste. Therefore, an integrated vascular network is required for clinical translation of engineered tissues. Microvessels have been described as <150 μm in diameter, but clinically they are defined as <1 mm. With new advances in super microsurgery, vessels less than 1 mm can be anastomosed to the recipient circulation. However, this technical advancement still relies on the creation of a stable engineered microcirculation that is amenable to surgical manipulation and is readily perfusable. Microvascular engineering lays on the crossroads of microfabrication, microfluidics, and tissue engineering strategies that utilize various cellular constituents. Early research focused on vascularization by co-culture and cellular interactions, with the addition of angiogenic growth factors to promote vascular growth. Since then, multiple strategies have been utilized taking advantage of innovations in additive manufacturing, biomaterials, and cell biology. However, the anatomy and dynamics of native blood vessels has not been consistently replicated. Inconsistent results can be partially attributed to cell sourcing which remains an enigma for microvascular engineering. Variations of endothelial cells, endothelial progenitor cells, and stem cells have all been used for microvascular network fabrication along with various mural cells. As each source offers advantages and disadvantages, there continues to be a lack of consensus. Furthermore, discord may be attributed to incomplete understanding about cell isolation and characterization without considering the microvascular architecture of the desired tissue/organ.
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Wang K, Man K, Liu J, Liu Y, Chen Q, Zhou Y, Yang Y. Microphysiological Systems: Design, Fabrication, and Applications. ACS Biomater Sci Eng 2020; 6:3231-3257. [PMID: 33204830 PMCID: PMC7668566 DOI: 10.1021/acsbiomaterials.9b01667] [Citation(s) in RCA: 38] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Microphysiological systems, including organoids, 3-D printed tissue constructs and organ-on-a-chips (organ chips), are physiologically relevant in vitro models and have experienced explosive growth in the past decades. Different from conventional, tissue culture plastic-based in vitro models or animal models, microphysiological systems recapitulate key microenvironmental characteristics of human organs and mimic their primary functions. The advent of microphysiological systems is attributed to evolving biomaterials, micro-/nanotechnologies and stem cell biology, which enable the precise control over the matrix properties and the interactions between cells, tissues and organs in physiological conditions. As such, microphysiological systems have been developed to model a broad spectrum of organs from microvasculature, eye, to lung and many others to understand human organ development and disease pathology and facilitate drug discovery. Multiorgans-on-a-chip systems have also been developed by integrating multiple associated organ chips in a single platform, which allows to study and employ the organ function in a systematic approach. Here we first discuss the design principles of microphysiological systems with a focus on the anatomy and physiology of organs, and then review the commonly used fabrication techniques and biomaterials for microphysiological systems. Subsequently, we discuss the recent development of microphysiological systems, and provide our perspectives on advancing microphysiological systems for preclinical investigation and drug discovery of human disease.
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Affiliation(s)
- Kai Wang
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Kun Man
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Jiafeng Liu
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
| | - Yang Liu
- North Texas Eye Research Institute, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107, United States
| | - Qi Chen
- The Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China
| | - Yong Zhou
- Department of Emergency, Xinqiao Hospital, Chongqing 400037, China
| | - Yong Yang
- Department of Biomedical Engineering, University of North Texas, Denton, Texas 76207, United States
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Alghamdi AAA, Benwell CJ, Atkinson SJ, Lambert J, Johnson RT, Robinson SD. NRP2 as an Emerging Angiogenic Player; Promoting Endothelial Cell Adhesion and Migration by Regulating Recycling of α5 Integrin. Front Cell Dev Biol 2020; 8:395. [PMID: 32528960 PMCID: PMC7264094 DOI: 10.3389/fcell.2020.00395] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Accepted: 04/29/2020] [Indexed: 01/01/2023] Open
Abstract
Angiogenesis relies on the ability of endothelial cells (ECs) to migrate over the extracellular matrix via integrin receptors to respond to an angiogenic stimulus. Of the two neuropilin (NRP) orthologs to be identified, both have been reported to be expressed on normal blood and lymphatic ECs, and to play roles in the formation of blood and lymphatic vascular networks during angiogenesis. Whilst the role of NRP1 and its interactions with integrins during angiogenesis has been widely studied, the role of NRP2 in ECs is poorly understood. Here we demonstrate that NRP2 promotes Rac-1 mediated EC adhesion and migration over fibronectin (FN) matrices in a mechanistically distinct fashion to NRP1, showing no dependence on β3 integrin (ITGB3) expression, or VEGF stimulation. Furthermore, we highlight evidence of a regulatory crosstalk between NRP2 and α5 integrin (ITGA5) in ECs, with NRP2 depletion eliciting an upregulation of ITGA5 expression and disruptions in ITGA5 cellular organization. Finally, we propose a mechanism whereby NRP2 promotes ITGA5 recycling in ECs; NRP2 depleted ECs were found to exhibit reduced levels of total ITGA5 subunit recycling compared to wild-type (WT) ECs. Our findings expose NRP2 as a novel angiogenic player by promoting ITGA5-mediated EC adhesion and migration on FN.
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Affiliation(s)
- Abdullah A A Alghamdi
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
| | - Christopher J Benwell
- Gut Microbes and Health, Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom
| | - Samuel J Atkinson
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
| | - Jordi Lambert
- Faculty of Medicine and Health Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
| | - Robert T Johnson
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom
| | - Stephen D Robinson
- School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, United Kingdom.,Gut Microbes and Health, Quadram Institute Bioscience, Norwich Research Park, Norwich, United Kingdom
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Aoki H, Yamashita M, Hashita T, Ogami K, Hoshino S, Iwao T, Matsunaga T. Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-β, and GSK3β. Heliyon 2020; 6:e03493. [PMID: 32154424 PMCID: PMC7056658 DOI: 10.1016/j.heliyon.2020.e03493] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2019] [Revised: 02/14/2020] [Accepted: 02/24/2020] [Indexed: 01/29/2023] Open
Abstract
Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of differentiated cells from hiPSCs, purification via an antigen–antibody reaction, which damages the cells, is indispensable. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83–01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-β]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3β [GSK3β] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.
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Affiliation(s)
- Hiromasa Aoki
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Misaki Yamashita
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Tadahiro Hashita
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Koichi Ogami
- Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Shinichi Hoshino
- Department of Biological Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Takahiro Iwao
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
| | - Tamihide Matsunaga
- Department of Clinical Pharmacy, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, 467-8603, Japan
- Corresponding author.
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Von Willebrand Disease: From In Vivo to In Vitro Disease Models. Hemasphere 2020; 3:e297. [PMID: 31942548 PMCID: PMC6919471 DOI: 10.1097/hs9.0000000000000297] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2019] [Accepted: 09/04/2019] [Indexed: 01/28/2023] Open
Abstract
Von Willebrand factor (VWF) plays an essential role in primary hemostasis and is exclusively synthesized and stored in endothelial cells and megakaryocytes. Upon vascular injury, VWF is released into the circulation where this multimeric protein is required for platelet adhesion. Defects of VWF lead to the most common inherited bleeding disorder von Willebrand disease (VWD). Three different types of VWD exist, presenting with varying degrees of bleeding tendencies. The pathophysiology of VWD can be investigated by examining the synthesis, storage and secretion in VWF producing cells. These cells can either be primary VWF producing cells or transfected heterologous cell models. For many years transfected heterologous cells have been used successfully to elucidate many aspects of VWF synthesis. However, those cells do not fully reflect the characteristics of primary cells. Obtaining primary endothelial cells or megakaryocytes with a VWD phenotype, requires invasive procedures, such as vessel collection or a bone marrow biopsy. A more recent and promising development is the isolation of endothelial colony forming cells (ECFCs) from peripheral blood as a true-to-nature cell model. Alternatively, various animal models are available but limiting, therefore, new approaches are needed to study VWD and other bleeding disorders. A potential versatile source of endothelial cells and megakaryocytes could be induced pluripotent stem cells (iPSCs). This review gives an overview of models that are available to study VWD and VWF and will discuss novel approaches that can be considered to improve the understanding of the structural and functional mechanisms underlying this disease.
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Binding and Structural Properties of DNA Aptamers with VEGF-A-Mimic Activity. MOLECULAR THERAPY. NUCLEIC ACIDS 2020; 19:1145-1152. [PMID: 32059340 PMCID: PMC7016029 DOI: 10.1016/j.omtn.2019.12.034] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/20/2019] [Revised: 12/04/2019] [Accepted: 12/23/2019] [Indexed: 12/27/2022]
Abstract
Vascular endothelial growth factors (VEGFs) are hypoxia-inducible secreted proteins to promote angiogenesis, in which VEGF-A is an important molecule that binds and activates VEGF receptor-1 (VEGFR-1) and VEGFR-2. In this study, two DNA aptamers, Apt01 and Apt02, were successfully isolated by alternating consecutive systematic evolution of ligands by exponential enrichment (SELEX) against VEGFR-1 and -2 using deep sequencing analysis in an early selection round. Their binding affinities for VEGFR-2 were lower than that of VEGFR-1, which is similar to that of VEGF-A. Structural analyses with the measurements of circular dichroism spectra and ultraviolet melting curve showed that Apt01 possessed the stem-loop structure in the molecule, whereas Apt02 formed G-quadruplex structures. In addition, Apt02 accelerated a tube formation of human umbilical vein endothelial cells faster than Apt01, which was affected by difference of binding affinity and nuclease resistance due to G-quadruplex structures. These results demonstrated that Apt02 might have a potential to function as an alternative to VEGF-A.
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Shear Stress Promotes Arterial Endothelium-Oriented Differentiation of Mouse-Induced Pluripotent Stem Cells. Stem Cells Int 2019; 2019:1847098. [PMID: 31827524 PMCID: PMC6881757 DOI: 10.1155/2019/1847098] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2019] [Revised: 09/05/2019] [Accepted: 10/17/2019] [Indexed: 12/29/2022] Open
Abstract
Establishment of a functional vascular network, which is required in tissue repair and regeneration, needs large-scale production of specific arterial or venous endothelial cells (ECs) from stem cells. Previous in vitro studies by us and others revealed that shear stress induces EC differentiation of bone marrow-derived mesenchymal stem cells and embryonic stem cells. In this study, we focused on the impact of different magnitudes of shear stress on the differentiation of mouse-induced pluripotent stem cells (iPSCs) towards arterial or venous ECs. When iPSCs were exposed to shear stress (5, 10, and 15 dyne/cm2) with 50 ng/mL vascular endothelial growth factor and 10 ng/mL fibroblast growth factor, the expression levels of the general EC markers and the arterial markers increased, and the stress amplitude of 10 dyne/cm2 could be regarded as a proper promoter, whereas the venous and lymphatic markers had little or no expression. Further, shear stress caused cells to align parallel to the direction of the flow, induced cells forming functional tubes, and increased the secretion of nitric oxide. In addition, Notch1 was significantly upregulated, and the Notch ligand Delta-like 4 was activated in response to shear stress, while inhibition of Notch signaling by DAPT remarkably abolished the shear stress-induced arterial epithelium differentiation. Taken together, our results indicate that exposure to appropriate shear stress facilitated the differentiation of mouse iPSCs towards arterial ECs via Notch signaling pathways, which have potential applications for both disease modeling and regenerative medicine.
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Sex-dependent VEGF expression underlies variations in human pluripotent stem cell to endothelial progenitor differentiation. Sci Rep 2019; 9:16696. [PMID: 31723192 PMCID: PMC6853961 DOI: 10.1038/s41598-019-53054-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2019] [Accepted: 10/28/2019] [Indexed: 12/21/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and a high proliferative capacity. To realize this potential, development of efficient hPSC differentiation protocols is required. In this work, sex-based differences are identified in a GSK3 inhibitor based endothelial progenitor differentiation protocol. While male hPSCs efficiently differentiate into CD34 + CD31+ endothelial progenitors upon GSK3 inhibition, female hPSCs showed limited differentiation capacity using this protocol. Using VE-cadherin-GFP knockin reporter cells, female cells showed significantly increased differentiation efficiency when treated with VEGF during the second stage of endothelial progenitor differentiation. Interestingly, male cells showed no significant change in differentiation efficiency with VEGF treatment, but did show augmented early activation of VE-cadherin expression. A sex-based difference in endogenous expression of VEGF was identified that is likely the underlying cause of discrepancies in sex-dependent differentiation efficiency. These findings highlight the importance of sex differences in progenitor biology and the development of new stem cell differentiation protocols.
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Identification of Cardiomyocyte-Fated Progenitors from Human-Induced Pluripotent Stem Cells Marked with CD82. Cell Rep 2019; 22:546-556. [PMID: 29320747 DOI: 10.1016/j.celrep.2017.12.057] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2017] [Revised: 10/22/2017] [Accepted: 12/17/2017] [Indexed: 02/06/2023] Open
Abstract
Here, we find that human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM)-fated progenitors (CFPs) that express a tetraspanin family glycoprotein, CD82, almost exclusively differentiate into CMs both in vitro and in vivo. CD82 is transiently expressed in late-stage mesoderm cells during hiPSC differentiation. Purified CD82+ cells gave rise to CMs under nonspecific in vitro culture conditions with serum, as well as in vivo after transplantation to the subrenal space or injured hearts in mice, indicating that CD82 successfully marks CFPs. CD82 overexpression in mesoderm cells as well as in undifferentiated hiPSCs increased the secretion of exosomes containing β-catenin and reduced nuclear β-catenin protein, suggesting that CD82 is involved in fated restriction to CMs through Wnt signaling inhibition. This study may contribute to the understanding of CM differentiation mechanisms and to cardiac regeneration strategies.
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Arora S, Yim EKF, Toh YC. Environmental Specification of Pluripotent Stem Cell Derived Endothelial Cells Toward Arterial and Venous Subtypes. Front Bioeng Biotechnol 2019; 7:143. [PMID: 31259171 PMCID: PMC6587665 DOI: 10.3389/fbioe.2019.00143] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2019] [Accepted: 05/28/2019] [Indexed: 12/25/2022] Open
Abstract
Endothelial cells (ECs) are required for a multitude of cardiovascular clinical applications, such as revascularization of ischemic tissues or endothelialization of tissue engineered grafts. Patient derived primary ECs are limited in number, have donor variabilities and their in vitro phenotypes and functions can deteriorate over time. This necessitates the exploration of alternative EC sources. Although there has been a recent surge in the use of pluripotent stem cell derived endothelial cells (PSC-ECs) for various cardiovascular clinical applications, current differentiation protocols yield a heterogeneous EC population, where their specification into arterial or venous subtypes is undefined. Since arterial and venous ECs are phenotypically and functionally different, inappropriate matching of exogenous ECs to host sites can potentially affect clinical efficacy, as exemplified by venous graft mismatch when placed into an arterial environment. Therefore, there is a need to design and employ environmental cues that can effectively modulate PSC-ECs into a more homogeneous arterial or venous phenotype for better adaptation to the host environment, which will in turn contribute to better application efficacy. In this review, we will first give an overview of the developmental and functional differences between arterial and venous ECs. This provides the foundation for our subsequent discussion on the different bioengineering strategies that have been investigated to varying extent in providing biochemical and biophysical environmental cues to mature PSC-ECs into arterial or venous subtypes. The ability to efficiently leverage on a combination of biochemical and biophysical environmental cues to modulate intrinsic arterio-venous specification programs in ECs will greatly facilitate future translational applications of PSC-ECs. Since the development and maintenance of arterial and venous ECs in vivo occur in disparate physio-chemical microenvironments, it is conceivable that the application of these environmental factors in customized combinations or magnitudes can be used to selectively mature PSC-ECs into an arterial or venous subtype.
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Affiliation(s)
- Seep Arora
- Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore.,Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, Singapore, Singapore
| | - Evelyn K F Yim
- Department of Chemical Engineering, University of Waterloo, Waterloo, ON, Canada
| | - Yi-Chin Toh
- Department of Biomedical Engineering, National University of Singapore, Singapore, Singapore.,Singapore Institute for Neurotechnology (SINAPSE), National University of Singapore, Singapore, Singapore.,Biomedical Institute for Global Health Research and Technology (BIGHEART), National University of Singapore, Singapore, Singapore.,NUS Tissue Engineering Program, National University of Singapore, Singapore, Singapore
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Zhang J, Zhang CF, Li QL, Chu CH. Cyclic Adenosine Monophosphate Promotes Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via Suppression of Transforming Growth Factor Beta 1 Signaling. J Endod 2019; 45:150-155. [PMID: 30711170 DOI: 10.1016/j.joen.2018.10.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Revised: 08/14/2018] [Accepted: 10/18/2018] [Indexed: 01/09/2023]
Abstract
INTRODUCTION Stem cells from the apical papilla (SCAPs) possess strong odonto/osteogenic differentiation potential. This study investigated the effect of cyclic adenosine monophosphate (cAMP) on odonto/osteogenic differentiation of SCAPs and the underlining interplay between cAMP and transforming growth factor beta 1 (TGF-β1). METHODS SCAPs were stimulated with an activator of cAMP (forskolin) in the presence of either TGF-β1 or a TGF-β1 inhibitor. The amounts of calcium mineral deposition and alkaline phosphatase activity were determined. Quantitative real-time polymerase chain reaction was performed to elucidate cAMP on the TGF-β1-mediated odonto/osteogenic differentiation of SCAPs. The effect of cAMP on the phosphorylation of Smad2/Smad3 and extracellular-regulated kinase (ERK)/P38 induced by TGF-β1 was analyzed by Western blotting. RESULTS Cotreatment with forskolin and a TGF-β1 inhibitor enhanced alkaline phosphatase activity and deposition of calcium minerals in SCAPs. Moreover, the TGF-β1 inhibitor synergized the effect of forskolin on the expression of type I collagen and runt-related transcription factor 2. The results of Western blotting revealed that forskolin attenuated the unregulated expression of the phosphorylation of Smad3 and ERK induced by TGF-β1, and a cAMP inhibitor (H89) antagonized this effect. CONCLUSIONS This study showed that cAMP signaling exerts its up-regulating effects on the odonto/osteogenic differentiation of SCAPs by interfering with TGF-β1 signaling via inhibiting Smad3 and ERK phosphorylation.
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Affiliation(s)
- Jing Zhang
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China; Key Laboratory of Oral Disease Research of Anhui Province, Stomatological Hospital and College, Anhui Medical University, Hefei, China
| | - Cheng Fei Zhang
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China.
| | - Quan Li Li
- Key Laboratory of Oral Disease Research of Anhui Province, Stomatological Hospital and College, Anhui Medical University, Hefei, China
| | - Chun Hung Chu
- Faculty of Dentistry, The University of Hong Kong, Hong Kong, China
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Rosa S, Praça C, Pitrez PR, Gouveia PJ, Aranguren XL, Ricotti L, Ferreira LS. Functional characterization of iPSC-derived arterial- and venous-like endothelial cells. Sci Rep 2019; 9:3826. [PMID: 30846769 PMCID: PMC6405900 DOI: 10.1038/s41598-019-40417-9] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2018] [Accepted: 02/11/2019] [Indexed: 02/06/2023] Open
Abstract
The current work reports the functional characterization of human induced pluripotent stem cells (iPSCs)- arterial and venous-like endothelial cells (ECs), derived in chemically defined conditions, either in monoculture or seeded in a scaffold with mechanical properties similar to blood vessels. iPSC-derived arterial- and venous-like endothelial cells were obtained in two steps: differentiation of iPSCs into endothelial precursor cells (CD31pos/KDRpos/VE-Cadmed/EphB2neg/COUP-TFneg) followed by their differentiation into arterial and venous-like ECs using a high and low vascular endothelial growth factor (VEGF) concentration. Cells were characterized at gene, protein and functional levels. Functionally, both arterial and venous-like iPSC-derived ECs responded to vasoactive agonists such as thrombin and prostaglandin E2 (PGE2), similar to somatic ECs; however, arterial-like iPSC-derived ECs produced higher nitric oxide (NO) and elongation to shear stress than venous-like iPSC-derived ECs. Both cells adhered, proliferated and prevented platelet activation when seeded in poly(caprolactone) scaffolds. Interestingly, both iPSC-derived ECs cultured in monoculture or in a scaffold showed a different inflammatory profile than somatic ECs. Although both somatic and iPSC-derived ECs responded to tumor necrosis factor-α (TNF-α) by an increase in the expression of intercellular adhesion molecule 1 (ICAM-1), only somatic ECs showed an upregulation in the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1).
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Affiliation(s)
- S Rosa
- CNC UC- Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal
| | - C Praça
- CNC UC- Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal.,Faculty of Medicine, University of Coimbra, 3000-354, Coimbra, Portugal
| | - P R Pitrez
- CNC UC- Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal.,Faculty of Medicine, University of Coimbra, 3000-354, Coimbra, Portugal
| | - P José Gouveia
- CNC UC- Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal.,IIIUC- Institute for Interdisciplinary Research, University of Coimbra, Casa Costa Alemão - Pólo II, Rua Dom Francisco de Lemos, 3030-789, Coimbra, Portugal
| | - X L Aranguren
- Hematology and Cell Therapy Area, Clinica Universidad de Navarra, and Division of Oncology, Center for Applied Medical Research, University of Navarra, Pamplona, Spain
| | - L Ricotti
- The BioRobotics Institute, Scuola Superiore Sant' Anna, Viale Rinaldo Piaggio 34, 56025, Pontedera, PI, Italy
| | - L Silva Ferreira
- CNC UC- Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517, Coimbra, Portugal. .,Faculty of Medicine, University of Coimbra, 3000-354, Coimbra, Portugal.
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46
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Teng EL, Engler AJ. Mechanical influences on cardiovascular differentiation and disease modeling. Exp Cell Res 2019; 377:103-108. [PMID: 30794804 DOI: 10.1016/j.yexcr.2019.02.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2018] [Revised: 02/09/2019] [Accepted: 02/18/2019] [Indexed: 01/06/2023]
Abstract
Tissues are continuously exposed to forces in vivo, whether from fluid pressure in an artery from our blood or compressive forces on joints from our body weight. The forces that cells are exposed to arise almost immediately after conception; it is therefore important to understand how forces shape stem cell differentiation into lineage committed cells, how they help organize cells into tissues, and how forces can cause or exacerbate disease. No tissue is exempt, but cardiovascular tissues in particular are exposed to these forces. While animal models have been used extensively in the past, there is growing recognition of their limitations when modeling disease complexity or human genetics. In this mini review, we summarize current understanding of the mechanical influences on the differentiation of cardiovascular progeny, how the transduction of forces influence the onset of disease, and how engineering approaches applied to this problem have yielded systems that create mature-like human tissues in vitro in which to assess the impact of disease on cell function.
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Affiliation(s)
- Evan L Teng
- Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, United States; Sanford Consortium for Regenerative Medicine, La Jolla, CA 92037, United States
| | - Adam J Engler
- Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, United States; Sanford Consortium for Regenerative Medicine, La Jolla, CA 92037, United States.
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Meng F, Almohanna F, Altuhami A, Assiri AM, Broering D. Vasculature reconstruction of decellularized liver scaffolds via gelatin-based re-endothelialization. J Biomed Mater Res A 2019; 107:392-402. [PMID: 30508280 DOI: 10.1002/jbm.a.36551] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2018] [Revised: 08/08/2018] [Accepted: 08/29/2018] [Indexed: 12/15/2022]
Abstract
Decellularized liver scaffolds based liver engineering is a promising approach toward developing functional liver surrogates. However, a major obstacle to long-term transplantation is the hemocompatibility of the bioengineered liver surrogates. One approach to improve the hemocompatibility of engineered liver surrogates is re-endothelialization. In the current study, immortalized endothelial cells were perfused for re-endothelialization of decellularized rat liver scaffolds. When compared to the media-based perfusion approach, gelatin hydrogels-based perfusion significantly increased the number of cells that were retained in the decellularized liver scaffolds and the vascular lumen coverage ratio. Endothelial cells were lining along the vasculatures of the decellularized liver scaffolds and actively proliferating. Re-endothelialization improved the blood retention ability of the liver scaffold vasculatures. Doppler ultrasound detected active blood flows within the re-endothelialized liver scaffold transplants 8 days post-transplantation. Our results strengthened the feasibility of developing bioengineered liver surrogates utilizing decellularized liver scaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 392-402, 2019.
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Affiliation(s)
- Fanwei Meng
- Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
- Organ Transplantation Center, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
| | - Falah Almohanna
- Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
| | - Abdullah Altuhami
- Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
- Organ Transplantation Center, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
| | - Abdallah M Assiri
- Department of Comparative Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
- College of Medicine, AlFaisal University, Riyadh, 11211, Saudi Arabia
- Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, 34212, Saudi Arabia
| | - Dieter Broering
- Organ Transplantation Center, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia
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Nakhaei-Nejad M, Farhan M, Mojiri A, Jabbari H, Murray AG, Jahroudi N. Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells. Stem Cells 2019; 37:542-554. [PMID: 30682218 DOI: 10.1002/stem.2978] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2018] [Revised: 11/13/2018] [Accepted: 12/19/2018] [Indexed: 12/13/2022]
Abstract
Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542-554.
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Affiliation(s)
| | - Maikel Farhan
- Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada
| | - Anahita Mojiri
- Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Hosna Jabbari
- Department of Computer Science, University of Vermont, Burlington, Vermont, USA
| | - Allan G Murray
- Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Nadia Jahroudi
- Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
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49
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Bezenah JR, Rioja AY, Juliar B, Friend N, Putnam AJ. Assessing the ability of human endothelial cells derived from induced-pluripotent stem cells to form functional microvasculature in vivo. Biotechnol Bioeng 2019; 116:415-426. [PMID: 30414271 PMCID: PMC6322937 DOI: 10.1002/bit.26860] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 08/22/2018] [Accepted: 11/07/2018] [Indexed: 12/26/2022]
Abstract
Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell-based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced-pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising, potentially autologous, alternative cell source. However, it is unclear whether iPSC-ECs form the same robust microvasculature in vivo documented for other EC sources. In this study, we utilized a well-established in vivo model, in which ECs (iPSC-EC or human umbilical vein endothelial cells [HUVEC]) were coinjected with normal human lung fibroblasts (NHLFs) and a fibrin matrix into the dorsal flank of severe combined immunodeficiency mice to assess their ability to form functional microvasculature. Qualitatively, iPSC-ECs were capable of vessel formation and perfusion and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC-ECs exhibited a two-fold reduction in vessel density and a three-fold reduction in the number of perfused vessels compared with HUVECs. Further analysis revealed the presence of collagen-IV and α-smooth muscle actin were significantly lower around iPSC-EC/NHLF vasculature than in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these results demonstrate the need for increased iPSC-EC maturation for clinical translation to be realized.
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Affiliation(s)
- Jonathan R. Bezenah
- Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
| | - Ana Y. Rioja
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
| | - Benjamin Juliar
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
| | - Nicole Friend
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
| | - Andrew J. Putnam
- Department of Chemical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, USA 48109
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50
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Liu Y, Yamashita JK. AMPK activators contribute to maintain naïve pluripotency in mouse embryonic stem cells. Biochem Biophys Res Commun 2018; 509:24-31. [PMID: 30573360 DOI: 10.1016/j.bbrc.2018.11.164] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Accepted: 11/27/2018] [Indexed: 12/25/2022]
Abstract
Pluripotent stem cells retain the property to self-renew and differentiate into all cell types under defined conditions. Among mouse embryonic stem cells (ESCs), which are pluripotent but heterogenous in gene expression and morphology, an ESC population cultured in small molecule inhibitors of two kinases, MAPK/ERK kinase (Mek) and Glycogen synthase kinase 3 (Gsk3), and leukemia inhibitory factor (Lif) (2i/L) is considered to be naïve pluripotent with uniform pluripotent machinery operation. Though the gene regulatory mechanism for the naïve pluripotency has been investigated in recent years, it is still not fully elucidated. Here we show a novel signaling involved in the maintenance of naïve pluripotency. An AMP-activated protein kinase (AMPK) activator, AICAR (5-Aminoimidazole-4-carboxamied-1-β-riboside) blocked the differentiation of mouse naïve ESCs in the absence of 2i/L and maintained the naïve state. AICAR with Lif condition induced an almost comparable level of naïve pluripotent gene expression in mouse ESCs. Another AMPK activator, A769662, also showed similar effects. A p38 inhibitor, SB203580, blocked the AMPK activation-elicited naïve state maintenance. On the other hand, p38 activation partially mimicked the maintenance effects of AMPK activators, suggesting that p38 is one of the functional downstream molecules to conduct the AMPK effects. Thus, AMPK pathway should be involved in the molecular circuitry of naïve pluripotency in mouse ESCs. These findings would be a valuable clue to further elucidate the molecular machinery of naïve pluripotency.
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Affiliation(s)
- Yajing Liu
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan
| | - Jun K Yamashita
- The Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, 606-8507, Japan.
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