Culture Of hPSCs Research Articles

Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation

BackgroundHuman pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown.Methods and resultsSTBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs’ pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls.ConclusionsTogether, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Scalable expansion of human pluripotent stem cells under suspension culture condition with human platelet lysate supplementation.

The large-scale production of human pluripotent stem cells (hPSCs), including both embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), shows potential for advancing the translational realization of hPSC technology. Among multiple cell culture methods, suspension culture, also known as three-dimensional (3D) culture, stands out as a promising method to fulfill the large-scale production requirements. Under this 3D culture condition, cell expansion and the preservation of pluripotency and identity during long-term culture heavily relies on the culture medium. However, the xenogeneic supplements in culture medium remains an obstacle for the translation of cell and gene therapy applications from bench to bedside. Here, we tested human platelet lysate (hPL), a xeno-free and serum-free biological material, as a supplement in the 3D culture of hPSCs. We observed reduced intercellular variability and enhanced proliferation in both hESC and hiPSC lines. These cells, after extended culture in the hPL-supplemented system, maintained pluripotency marker expression, the capacity to differentiate into cells of all three germ layers, and normal karyotype, confirming the practicability and safety of hPL supplementation. Furthermore, through RNA-sequencing analysis, we found an upregulation of genes associated with cell cycle regulations in hPL-treated cells, consistent with the improved cellular division efficiency. Taken together, our findings underscore the potential of hPL as a xeno-free and serum-free supplement for the large-scale production of hPSCs, which holds promise for advancing clinical applications of these cells.

Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells

Cell expansion of human pluripotent stem cells (hPSCs) commonly depends on Matrigel as a coating matrix on two-dimensional (2D) culture plates and 3D microcarriers. However, the xenogenic Matrigel requires sophisticated quality-assurance processes to meet clinical requirements. In this study, we develop an innovative coating-free medium for expanding hPSCs. The xenofree medium supports the weekend-free culture and competitive growth of hPSCs on several cell culture plastics without an additional pre-coating process. The pluripotent stemness of the expanded cells is stably sustained for more than 10 passages, featured with high pluripotent marker expressions, normal karyotyping, and differentiating capacity for three germ layers. The expression levels of some integrins are reduced, compared with those of the hPSCs on Matrigel. This medium also successfully supports the clonal expansion and induced pluripotent stem cell establishment from mitochondrial-defective MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) patient’s peripheral blood mononuclear cells. This innovative hPSC medium provides a straightforward scale-up process for producing clinical-orientated hPSCs by excluding the conventional coating procedure.

Xeno-free culture and proliferation of hPSCs on 2D biomaterials.

Human pluripotent stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) have unlimited proliferative potential, whereas adult stem cells such as bone marrow-derived stem cells and adipose-derived stem cells have problems with aging. When hPSCs are intended to be cultured on feeder-free or xeno-free conditions without utilizing mouse embryonic fibroblasts or human fibroblasts, they cannot be cultured on conventional tissue culture polystyrene dishes, as adult stem cells can be cultured but should be cultivated on material surfaces grafted or coated with (a) natural or recombinant extracellular matrix (ECM) proteins, (b) ECM protein-derived peptides and specific synthetic polymer surfaces in xeno-free and/or chemically defined conditions. This review describes current developing cell culture biomaterials for the proliferation of hPSCs while maintaining the pluripotency and differentiation potential of the cells into 3 germ layers. Biomaterials for the cultivation of hPSCs without utilizing a feeder layer are essential to decrease the risk of xenogenic molecules, which contributes to the potential clinical usage of hPSCs. ECM proteins such as human recombinant vitronectin, laminin-511 and laminin-521 have been utilized instead of Matrigel for the feeder-free cultivation of hPSCs. The following biomaterials are also discussed for hPSC cultivation: (a) decellularized ECM, (b) peptide-grafted biomaterials derived from ECM proteins, (c) recombinant E-cadherin-coated surface, (d) polysaccharide-immobilized surface, (e) synthetic polymer surfaces with and without bioactive sites, (f) thermoresponsive polymer surfaces with and without bioactive sites, and (g) synthetic microfibrous scaffolds.

Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin.

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4β1 or αVβ1, but not α5β1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation.

Design of chemically defined synthetic substrate surfaces for the in vitro maintenance of human pluripotent stem cells: A review.

Human pluripotent stem cells (hPSCs) have the potential of long-term self-renewal and differentiation into nearly all cell types in vitro. Prior to the downstream applications, the design of chemically defined synthetic substrates for the large-scale proliferation of quality-controlled hPSCs is critical. Although great achievements have been made, Matrigel and recombinant proteins are still widely used in the fundamental research and clinical applications. Therefore, much effort is still needed to improve the performance of synthetic substrates in the culture of hPSCs, realizing their commercial applications. In this review, we summarized the design of reported synthetic substrates and especially their limitations in terms of cell culture. Moreover, much attention was paid to the development of promising peptide displaying surfaces. Besides, the biophysical regulation of synthetic substrate surfaces as well as the three-dimensional culture systems were described.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture.

ObjectivesFor clinical applications of cell‐based therapies, a large quantity of human pluripotent stem cells (hPSCs) produced in standardized and scalable culture processes is required. Currently, microcarrier‐free suspension culture shows potential for large‐scale expansion of hPSCs; however, hPSCs tend to aggregate during culturing leading to a negative effect on cell yield. To overcome this problem, we developed a novel protocol to effectively control the sizes of cell aggregates and enhance the cell proliferation during the expansion of hPSCs in suspension.Materials and MethodshPSCs were expanded in suspension culture supplemented with polyvinyl alcohol (PVA) and dextran sulphate (DS), and 3D suspension culture of hPSCs formed cell aggregates under static or dynamic conditions. The sizes of cell aggregates and the cell proliferation as well as the pluripotency of hPSCs after expansion were assessed using cell counting, size analysis, real‐time quantitative polymerase chain reaction, flow cytometry analysis, immunofluorescence staining, embryoid body formation, teratoma formation and transcriptome sequencing.ResultsOur results demonstrated that the addition of DS alone effectively prevented hPSC aggregation, while the addition of PVA significantly enhanced hPSC proliferation. The combination of PVA and DS not only promoted cell proliferation of hPSCs but also produced uniform and size‐controlled cell aggregates. Moreover, hPSCs treated with PVA, or DS or a combination, maintained the pluripotency and were capable of differentiating into all three germ layers. mRNA‐seq analysis demonstrated that the combination of PVA and DS significantly promoted hPSC proliferation and prevented cell aggregation through improving energy metabolism‐related processes, regulating cell growth, cell proliferation and cell division, as well as reducing the adhesion among hPSC aggregates by affecting expression of genes related to cell adhesion.ConclusionsOur results represent a significant step towards developing a simple and robust approach for the expansion of hPSCs in large scale.

Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells.

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

Engineered peptide modified hydrogel platform for propagation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine, however these cells require expansion in cell colonies to maintain cell-cell contact, thus limiting the scalability needed to meet the demands of cell therapy. While the use of a Rho-associated protein kinase (ROCK) inhibitor will allow for culture of single cell hPSCs, typically only 50% of cells are recovered after dissociation. When hPSCs lose cell-cell contact through E-cadherin, dissociation induced apoptosis occurs. In this study, we hypothesized that the extracellular E-cadherin domain of hPSCs will bind to synthetic E-cadherin peptides presented on a hydrogel substrate, mimicking the required cell-cell contact and thereby retaining single-cell viability and clonogenicity. Hence, the objective of this study was to functionalize alginate hydrogels with synthetic peptides mimicking E-cadherin and evaluate peptide performance in promoting cell attachment, viability, maintaining pluripotency, and differentiation potential. We observed that alginate conjugated with synthetic E-cadherin peptides not only supported initial cell attachment with high viability, but also supported hPSC propagation and high fold expansion. hPSCs propagated on the peptide modified substrates maintained the hPSC characteristic pluripotency and differentiation potential, characterized by both spontaneous and directed differentiation. Statement of SignificanceHuman pluripotent stem cells (hPSCs) have enormous potential to alleviate cell needs for regenerative medicine and cell therapy. However, scalable culture of hPSCs is challenged by its need for maintenance of cell-cell contact, dissociation of which triggers the apoptotic pathway. Hence hPSCs are commonly maintained as colonies over Matrigel coated culture plates. Furthermore, use of xenogenic and undefined Matrigel compromises the translational potential of hPSCs. In this work we have developed a completely defined substrate to enable adherent culture of hPSCs as single cells. This substrate prevents apoptosis of the single cells and allows significant fold expansion of hPSCs while maintaining pluripotency and differentiation potential. The developed substrate is expected to be a cost-effective and translatable alternative to Matrigel.

The immobilization of fibronectin- and fibroblast growth factor 2-derived peptides on a culture plate supports the attachment and proliferation of human pluripotent stem cells.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.

Human Caesarean scar-derived feeder cells: a novel feeder cell type for culturing human pluripotent stem cells without exogenous basic fibroblast growth factor supplementation.

In a feeder-dependent culture system of human pluripotent stem cells (hPSCs), coculture with mouse embryonic fibroblasts may limit the clinical use of hPSCs. The aim of this study was to determine the feasibility of using human Caesarean scar fibroblasts (HSFs) as feeder cells for the culture of hPSCs. HSFs were isolated and characterised and cocultured with hPSCs, and the pluripotency, differentiation ability and karyotypic stability of hPSCs were determined. Inactivated HSFs expressed genes (including inhibin subunit beta A (INHBA), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), transforming growth factor-β1 (TGFB1), collagen alpha-1(I) (COL1A1) and fibronectin-1 (FN1) that have been implicated in the maintenance of hPSC pluripotency. When HSFs were used as feeder cells, the pluripotency and karyotypic stability of hPSC lines did not change after prolonged coculture. Interestingly, exogenous FGF2 could be omitted from the culture medium when HSFs were used as feeder cells for hESCs but not hiPSCs. hESCs cocultured with HSF feeder cells in medium without FGF2 supplementation maintained their pluripotency (as confirmed by the expression of pluripotency markers and genes), differentiated invitro into embryonic germ layers and maintained their normal karyotype. The present study demonstrates that HSFs are a novel feeder cell type for culturing hPSCs and that supplementation of exogenous FGF2 is not necessary for the Chula2.hES line.

Human Hematopoietic Stem Cells: Concepts and Perspectives on the Biology and Use of Fresh Versus In Vitro–Generated Cells for Therapeutic Applications

The in vitro production of human hematopoietic stem cells (hHSCs) has caused great interest due to its clinical impact. The main goal of the present article is to review the information existing today on the in vitro generation of hHSCs and their molecular and functional integrity as compared with fresh hHSCs. By using different in vitro systems, hHSCs have been generated from fresh hHSCs (obtained from bone marrow, peripheral blood, or cord blood) and from pluripotent stem cells. Although functional and molecular gaps have been observed between fresh and in vitro–derived hHSCs, recent clinical trials indicate that hHSCs generated in vitro from cord blood are capable of long-term hematopoietic reconstitution in transplanted patients. To date, no data exist on the clinical use of hHSCs derived from human pluripotent stem cells (hPSCs). Significant achievements in hHSC expansion and manipulation, as well as in the culture and differentiation of hPSCs, have been reported. All this, together with innovative clinical trials for the treatment of hematologic disorders, will be fundamental for the in vitro generation of hHSCs and their application in clinical settings.

Expansion Culture of Human Pluripotent Stem Cells and Production of Cardiomyocytes.

Transplantation of human pluripotent stem cell (hPSCs)-derived cardiomyocytes for the treatment of heart failure is a promising therapy. In order to implement this therapy requiring numerous cardiomyocytes, substantial production of hPSCs followed by cardiac differentiation seems practical. Conventional methods of culturing hPSCs involve using a 2D culture monolayer that hinders the expansion of hPSCs, thereby limiting their productivity. Advanced culture of hPSCs in 3D aggregates in the suspension overcomes the limitations of 2D culture and attracts immense attention. Although the hPSC production needs to be suitable for subsequent cardiac differentiation, many studies have independently focused on either expansion of hPSCs or cardiac differentiation protocols. In this review, we summarize the recent approaches to expand hPSCs in combination with cardiomyocyte differentiation. A comparison of various suspension culture methods and future prospects for dynamic culture of hPSCs are discussed in this study. Understanding hPSC characteristics in different models of dynamic culture helps to produce numerous cells that are useful for further clinical applications.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

Scalable and physiologically relevant microenvironments for human pluripotent stem cell expansion and differentiation

Human pluripotent stem cells (hPSCs) are required in large numbers for various biomedical applications. However, the scalable and cost-effective culturing of high quality hPSCs and their derivatives remains very challenging. Here, we report a novel and physiologically relevant 3D culture system (called the AlgTube cell culture system) for hPSC expansion and differentiation. With this system, cells are processed into and cultured in microscale alginate hydrogel tubes that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses in the culture vessel and limit the cell mass smaller than 400 μm in diameter to ensure efficient mass transport, creating cell-friendly microenvironments for growing cells. This system is simple, scalable, highly efficient, defined and compatible with the current good manufacturing practices. Under optimized culture conditions, the AlgTubes enabled long-term culture of hPSCs (>10 passages, >50 days) with high cell viability, high growth rate (1000-fold expansion over 10 days per passage), high purity (>95% Oct4+) and high yield (5.0 × 108 cells ml−1), all of which offer considerable advantages compared to current approaches. Moreover, the AlgTubes enabled directed differentiation of hPSCs into various tissue cells. This system can be readily scaled to support research from basic biological study to clinical development and the future industry-scale production.

Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture.

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc.

Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner

We describe highly effective adhesion culture of human pluripotent stem cells (hPSCs) using laminin fragments without precoating. Culture substrates have been generally thought to exert a cell adhesion effect when they are precoated onto culture vessels. However, simple addition of laminin fragments to a cell suspension during passaging accelerated the adhesion of single dissociated hPSCs onto culture vessels that were not precoated with any culture substrate. Interestingly, similar to conventional precoating, the uncoated addition of laminin fragments supported robust adhesion of single hPSCs and maximum adhesion at a much lower concentration compared with precoating. Similar to precoating laminin fragments, hPSCs seeded with uncoated laminin fragments grew well without cell detachment and maintained pluripotency after continuous subculture. We tested other culture substrates, including full-length laminin and vitronectin, to support hPSC adhesion in the uncoated manner, but only laminin fragments had the potential for application in the uncoated manner. This cost-effective and time-efficient method may contribute to expansion of culture of hPSCs and accelerate the development of regenerative medicine using hPSCs.

Peptide-Decorated Nanofibrous Niche Augments In Vitro Directed Osteogenic Conversion of Human Pluripotent Stem Cells.

Realization of clinical potential of human pluripotent stem cells (hPSCs) in bone regenerative medicine requires development of simple and safe biomaterials for expansion of hPSCs followed by directing their lineage commitment to osteoblasts. In the present study, a chemically defined peptide-decorated polycaprolactone (PCL) nanofibrous microenvironment was prepared through electrospinning technology and subsequent conjugation with vitronectin peptide to promote the culture and osteogenic potential of hPSCs in vitro. The results indicated that hPSCs successfully proliferated and maintained their pluripotency on the biointerface of peptide-conjugated nanofibers without Matrigel under defined conditions. Moreover, the prepared niche exhibited an appealing ability in promoting directed differentiation of hPSCs to osteoblastic phenotype without embryoid body formation step, determined from the cell morphological alteration, alkaline phosphate activity, and osteogenesis-related gene expression, as well as protein production. Such well-defined, xeno-free, and safe nanofiber scaffolds that allow the survival and facilitate osteo-differentiation of hPSCs provide a novel platform for hPSCs differentiation via cell-nanofiber interplay, and possess great value in accelerating the translational perspectives of hPSCs in bone tissue engineering.

Culture, Adaptation, and Expansion of Pluripotent Stem Cells.

The ability of human pluripotent stem cells (hPSCs) to proliferate indefinitely in culture while maintaining their pluripotent properties makes them a powerful tool for use in research, and provides tremendous potential for diagnostic testing, and therapeutic application. Success in these areas, however, is dependent on the ability to effectively expand them in long-term culture while preserving their distinct nature. Contained in this chapter are detailed protocols for the feeder-independent culture and expansion of hPSCs using mTeSR1 medium and Matrigel matrix, and guidelines for the successful transfer of those cells to alternative platforms. These protocols have been used widely by laboratories around the world to successfully expand hPSCs for long-term culture while maintaining their undifferentiated, pluripotent state.