Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity

Higuchi, Akon; Kao, Shih Hsuan; Ling, Qing; Chen, Yen-Ming; Li, Hsing Fen; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Murugan, Kadarkarai; Chang, Shih Chang; Lee, Hsin Chung; Hsu, Shih Tien; Kumar, Suresh; Umezawa, Akihiro

doi:10.1038/srep18136

Cited by 59 publications

(82 citation statements)

References 57 publications

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“…Another study developed synthetic substrates displaying heparin-binding peptides, which can interact with cell-surface glycosaminoglycans, showing that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells [ 48 ]. Another study developed polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time [ 49 ]. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions.…”

Section: Synthetic Substratesmentioning

confidence: 99%

Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

Hayashi

Furue

2016

Stem Cells International

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In recent years, as human pluripotent stem cells (hPSCs) have been commonly cultured in feeder-free conditions, a number of cell culture substrates have been applied or developed. However, the functional roles of these substrates in maintaining hPSC self-renewal remain unclear. Here in this review, we summarize the types of these substrates and their effect on maintaining hPSC self-renewal. Endogenous extracellular matrix (ECM) protein expression has been shown to be crucial in maintaining hPSC self-renewal. These ECM molecules interact with integrin cell-surface receptors and transmit their cellular signaling. We discuss the possible effect of integrin-mediated signaling pathways on maintaining hPSC self-renewal. Activation of integrin-linked kinase (ILK), which transmits ECM-integrin signaling to AKT (also known as protein kinase B), has been shown to be critical in maintaining hPSC self-renewal. Also, since naïve pluripotency has been widely recognized as an alternative pluripotent state of hPSCs, we discuss the possible effects of culture substrates and integrin signaling on naïve hPSCs based on the studies of mouse embryonic stem cells. Understanding the role of culture substrates in hPSC self-renewal and differentiation enables us to control hPSC behavior precisely and to establish scalable or microfabricated culture technologies for regenerative medicine and drug development.

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Section: Synthetic Substratesmentioning

confidence: 99%

Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

Hayashi

Furue

2016

Stem Cells International

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“…For example, Synthemax (Corning), a synthetic peptide with vitronectin elements, is a GMP‐compatible product marketed for ESC culture and which has seen limited use in the generation of iPSCs . Synthetic hydrogel models for 3D culture of poly(N‐isopropylacrylamide)‐co‐poly(ethylene glycol; PNIPAAm‐PEG) and polyvinylalcohol‐co‐itaconic acid hydrogels with Synthemax have also been demonstrated . Another recently released product for cell therapy grade use is recombinant human laminin (Laminin‐521, BioLamina).…”

Section: Discussionmentioning

confidence: 99%

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

Gandhi

Knudsen

Hill

et al. 2019

Stem Cells Translational Medicine

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Human fibrin hydrogels are a popular choice for use as a biomaterial within tissue engineered constructs because they are biocompatible, nonxenogenic, autologous use compatible, and biodegradable. We have recently demonstrated the ability to culture induced pluripotent stem cell (iPSC)‐derived retinal pigment epithelium on fibrin hydrogels. However, iPSCs themselves have relatively few substrate options (e.g., laminin) for expansion in adherent cell culture for use in cell therapy. To address this, we investigated the potential of culturing iPSCs on fibrin hydrogels for three‐dimensional applications and further examined the use of fibrinogen, the soluble precursor protein, as a coating substrate for traditional adherent cell culture. iPSCs successfully adhered to and proliferated on fibrin hydrogels. The two‐dimensional culture with fibrinogen allows for immediate adaption of culture models to a nonxenogeneic model. Similarly, multiple commercially available iPSC lines adhered to and proliferated on fibrinogen coated surfaces. iPSCs cultured on fibrinogen expressed similar levels of the pluripotent stem cell markers SSea4 (98.7% ± 1.8%), Oct3/4 (97.3% ± 3.8%), TRA1‐60 (92.2% ± 5.3%), and NANOG (96.0% ± 3.9%) compared with iPSCs on Geltrex. Using a trilineage differentiation assay, we found no difference in the ability of iPSCs grown on fibrinogen or Geltrex to differentiate to endoderm, mesoderm, or ectoderm. Finally, we demonstrated the ability to differentiate iPSCs to endothelial cells using only fibrinogen coated plates. On the basis of these data, we conclude that human fibrinogen provides a readily available and inexpensive alternative to laminin‐based products for the growth, expansion, and differentiation of iPSCs for use in research and clinical cell therapy applications. stem cells translational medicine 2019;8:512–521

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“…By comparison, peptide-based methods, such as VDP, have the tremendous benefits of off-the-shelf availability and ease of use as these peptides can be coated onto tissue culture surfaces in a manner similar to ECMPs such as LN. As such, peptide-based materials have been used for the expansion and differentiation of hPSCs (66, 68). In particular, a similar peptide sequence that served for the basis for VDP in this study has been used for the long-term culture of undifferentiated hPSCs (27).…”

Section: Discussionmentioning

confidence: 99%

A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)

et al. 2017

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Despite therapeutic advances, neurodegenerative diseases and disorders remain some of the leading causes of mortality and morbidity in the United States. Therefore, cell-based therapies to replace lost or damaged neurons and supporting cells of the central nervous system (CNS) are of great therapeutic interest. To that end, human pluripotent stem cells (hPSC) derived neural progenitor cells (hNPCs) and their neuronal derivatives could provide the cellular ‘raw material’ needed for regenerative medicine therapies for a variety of CNS disorders. In addition, hNPCs derived from patient-specific hPSCs could be used to elucidate the underlying mechanisms of neurodegenerative diseases and identify potential drug candidates. However, the scientific and clinical application of hNPCs requires the development of robust, defined, and scalable substrates for their long-term expansion and neuronal differentiation. In this study, we rationally designed a vitronectin-derived peptide (VDP) that served as an adhesive growth substrate for the long-term expansion of several hNPC lines. Moreover, VDP-coated surfaces allowed for the directed neuronal differentiation of hNPC at levels similar to cells differentiated on traditional extracellular matrix protein-based substrates. Overall, the ability of VDP to support the long-term expansion and directed neuronal differentiation of hNPCs will significantly advance the future translational application of these cells in treating injuries, disorders, and diseases of the CNS.

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Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity

Cited by 59 publications

References 57 publications

Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

Biological Effects of Culture Substrates on Human Pluripotent Stem Cells

Human Fibrinogen for Maintenance and Differentiation of Induced Pluripotent Stem Cells in Two Dimensions and Three Dimensions

A robust vitronectin-derived peptide for the scalable long-term expansion and neuronal differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs)

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