Expansion Of hPSCs Research Articles

A retinoid analogue, TTNPB, promotes clonal expansion of human pluripotent stem cells by upregulating CLDN2 and HoxA1

Enzymatic dissociation of human pluripotent stem cells (hPSCs) into single cells during routine passage leads to massive cell death. Although the Rho-associated protein kinase inhibitor, Y-27632 can enhance hPSC survival and proliferation at high seeding density, dissociated single cells undergo apoptosis at clonal density. This presents a major hurdle when deriving genetically modified hPSC lines since transfection and genome editing efficiencies are not satisfactory. As a result, colonies tend to contain heterogeneous mixtures of both modified and unmodified cells, making it difficult to isolate the desired clone buried within the colony. In this study, we report improved clonal expansion of hPSCs using a retinoic acid analogue, TTNPB. When combined with Y-27632, TTNPB synergistically increased hPSC cloning efficiency by more than 2 orders of magnitude (0.2% to 20%), whereas TTNPB itself increased more than double cell number expansion compared to Y-27632. Furthermore, TTNPB-treated cells showed two times higher aggregate formation and cell proliferation compared to Y-27632 in suspension culture. TTNPB-treated cells displayed a normal karyotype, pluripotency and were able to stochastically differentiate into all three germ layers both in vitro and in vivo. TTNBP acts, in part, by promoting cellular adhesion and self-renewal through the upregulation of Claudin 2 and HoxA1. By promoting clonal expansion, TTNPB provides a new approach for isolating and expanding pure hPSCs for future cell therapy applications.

IL-1β expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function

AbstractHematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1β (IL-1β) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1−/− mice show that TLR1/2-induced HSPC expansion is dependent on IL-1β signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1β and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1β expression.

Dextran sulfate prevents excess aggregation of human pluripotent stem cells in 3D culture by inhibiting ICAM1 expression coupled with down-regulating E-cadherin through activating the Wnt signaling pathway

BackgroundHuman pluripotent stem cells (hPSCs) have great potential in applications for regenerative medicine and drug development. However, 3D suspension culture systems for clinical-grade hPSC large-scale production have been a major challenge. Accumulating evidence has demonstrated that the addition of dextran sulfate (DS) could prevent excessive adhesion of hPSCs from forming larger aggregates in 3D suspension culture. However, the signaling and molecular mechanisms underlying this phenomenon remain elusive.MethodsBy using a cell aggregate culture assay and separating big and small aggregates in suspension culture systems, the potential mechanism and downstream target genes of DS were investigated by mRNA sequence analysis, qRT-PCR validation, colony formation assay, and interference assay.ResultsSince cellular adhesion molecules (CAMs) play important roles in hPSC adhesion and aggregation, we assumed that DS might prevent excess adhesion through affecting the expression of CAMs in hPSCs. As expected, after DS treatment, we found that the expression of CAMs was significantly down-regulated, especially E-cadherin (E-cad) and intercellular adhesion molecule 1 (ICAM1), two highly expressed CAMs in hPSCs. The role of E-cad in the adhesion of hPSCs has been widely investigated, but the function of ICAM1 in hPSCs is hardly understood. In the present study, we demonstrated that ICAM1 exhibited the capacity to promote the adhesion in hPSCs, and this adhesion was suppressed by the treatment with DS. Furthermore, transcriptomic analysis of RNA-seq revealed that DS treatment up-regulated genes related to Wnt signaling resulting in the activation of Wnt signaling in which SLUG, TWIST, and MMP3/7 were highly expressed, and further inhibited the expression of E-cad.ConclusionOur results demonstrated that DS played an important role in controlling the size of hPSC aggregates in 3D suspension culture by inhibiting the expression of ICAM1 coupled with the down-regulation of E-cad through the activation of the Wnt signaling pathway. These results represent a significant step toward developing the expansion of hPSCs under 3D suspension condition in large-scale cultures.

Colloidal Self-Assembled Patterns Maintain the Pluripotency and Promote the Hemopoietic Potential of Human Embryonic Stem Cells.

The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1–#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3–4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-β) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.

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.

Effects of Rigosertib (RIGO) Alone or in Combination with Azacitidine or Vorinostat on Epigenetic Reprogramming of CD34+ Cells in the Myelodysplastic Syndrome

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.RIG in combination with AZA or VOR (AZA/RIG, RIG/AZA, VOR/RIG or RIG/VOR) demonstrated differential effects on the association of RNA polymerase II (Pol II) with active histone marks (H3K4me3 and H3K4me2) in MDS-L and BW-90 cells. An overall decrease in association of Pol II/H3K4me2 was observed with the combinations (AZA/RIG, VOR/RIG or vice versa) compared to control in MDS-L and BW-90, 10-33% (ANOVA, p=0.0006), 9-20% (ANOVA, p=0.0004), respectively. Significant differences were observed in association of H3K4me3/Pol II in BW-90 cells (7-30%; ANOVA, p<0.0001). Similarly, in BM from a pt with MDS after 1 cycle of RIG and AZA treatment demonstrated a decrease in association of Pol II with H3K4me2 (67%) and H3K4me3 (28%).Treatment of MDS-L cells with RIG alone or in combination with AZA failed to induce expansion of CD34+ cells, but the RIG/VOR combination induced 1.9-fold expansion of CD34+ cells (ANOVA p=0.002). Individually AZA or VOR treatment led to an enhanced expansion of CD34+ cells, 3.8 and 4.0 fold, respectively (ANOVA, p=0.004). RIG alone or with AZA yielded maximum aldehyde dehydrogenase (ALDH) activity, a marker of primitive hematopoietic stem and progenitor cells (HSPCs) (ANOVA, p=0.006). However, a marked decrease in ALDH activity was observed in AZA or VOR or RIG/VOR, that was inversely proportion to the expansion of CD34+ cells.In a single MDS pt treated with RIG/AZA, expansion of primitive HPSCs expressing low levels of CD34 was observed. A highly expressed subpopulation of HPSCs, which co-existed prior to treatment of the pt and disappeared upon treatment, suggests either an induced cell death or commitment of HSPCs towards improved lineage differentiation in conjunction with clinical response.In order to evaluate effects of RIG on expansion of CD34+ cells, we examined the pluripotent genes (SOX2, OCT4 NANOG and ZIC3) in the BM from MDS pts, prior and after RIG/AZA treatment. RNA expression levels of SOX2, OCT4, NANOG and ZIC3 were elevated post treatment (≥2 fold). In MDS-L cells the pluripotency genes were highly upregulated in the presence of RIG or RIG/AZA or RIG/VOR (1.7-34 folds). These findings indicate that expression of pluripotency genes is a consequence of epigenetic reprogramming that favors expansion of more primitive HSPCs in a pt, while increase in ALDH activity was inversely proportional to the enhanced expansion of CD34+ cells in MDS-L suggesting a more primitive state of HSPC.The epigenetic events modulated by RIG in combination with either AZA or VOR led to global histone PTMs, differential Pol II association with active histone marks, epigenetic reprogramming of pluripotency genes and expansion of primitive HSPC with downregulation of the PI3K/AKT pathway and cell cycle checkpoint proteins. Collectively, these studies indicate that epigenetic effects of RIG on chromatin alterations lead to HSPC reprogramming that may lead to improved hematopoietic function and response in the clinical setting. These preclinical models suggest potential novel clinical strategies to improve outcomes for patients with higher-risk MDS. DisclosuresReddy:Onconova Therapeutics: Equity Ownership.

Discovery of a Novel Polymer for Xeno-Free, Long-Term Culture of Human Pluripotent Stem Cell Expansion.

Human pluripotent stem cells (hPSCs) can be expanded and differentiated in vitro into almost any adult tissue cell type, and thus have great potential as a source for cell therapies with biomedical application. In this study, a fully-defined polymer synthetic substrate is identified for hPSC culture in completely defined, xenogenic (xeno)-free conditions. This system can overcome the cost, scalability, and reproducibility limitations of current hPSC culture strategies, and facilitate large-scale production. A high-throughput, multi-generational polymer microarray platform approach is used to test over 600 unique polymers and rapidly assess hPSC-polymer interactions in combination with the fully defined xeno-free medium, Essential 8 (E8). This study identifies a novel nanoscale phase separated blend of poly(tricyclodecane-dimethanol diacrylate) and poly(butyl acrylate) (2:1 v/v), which supports long-term expansion of hPSCs and can be readily coated onto standard cultureware. Analysis of cell-polymer interface interactions through mass spectrometry and integrin blocking studies provides novel mechanistic insight into the role of the E8 proteins in promoting integrin-mediated hPSC attachment and maintaining hPSC signaling, including ability to undergo multi-lineage differentiation. This study therefore identifies a novel substrate for long-term serial passaging of hPSCs in serum-free, commercial chemically-defined E8, which provides a promising and economic hPSC expansion platform for clinical-scale application.

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 transcriptional regulator ZNF398 mediates pluripotency and epithelial character downstream of TGF-beta in human PSCs

Human pluripotent stem cells (hPSCs) have the capacity to give rise to all differentiated cells of the adult. TGF-beta is used routinely for expansion of conventional hPSCs as flat epithelial colonies expressing the transcription factors POU5F1/OCT4, NANOG, SOX2. Here we report a global analysis of the transcriptional programme controlled by TGF-beta followed by an unbiased gain-of-function screening in multiple hPSC lines to identify factors mediating TGF-beta activity. We identify a quartet of transcriptional regulators promoting hPSC self-renewal including ZNF398, a human-specific mediator of pluripotency and epithelial character in hPSCs. Mechanistically, ZNF398 binds active promoters and enhancers together with SMAD3 and the histone acetyltransferase EP300, enabling transcription of TGF-beta targets. In the context of somatic cell reprogramming, inhibition of ZNF398 abolishes activation of pluripotency and epithelial genes and colony formation. Our findings have clear implications for the generation of bona fide hPSCs for regenerative medicine.

Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells

Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. Additionally, the AIC medium shows a good compatibility for different hPSC lines under various culture conditions. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products.

End-to-End Platform for Human Pluripotent Stem Cell Manufacturing.

Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications.

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.

Enabling stem cell based therapies: end-to-end solution for human pluripotent stem cells manufacturing

Background & Aim Enabling stem cell-base therapies requires innovative solutions to close the gaps existing between research and commercialization. Allogeneic cell therapy indications that target large patient populations will necessitate the use of flexible cell production platforms to meet required cell quantities. Developing a truly scalable, controlled bioreactor platforms for cell expansion enables meeting cell quantity demand for clinical applications while allowing comparability between the various scales. Likewise, it enhances process automation and allows integration of online monitoring systems. The capability of effectively culturing adherent stem cells, namely pluripotent stem cells, will be presented. Methods, Results & Conclusion Our platform take advantage of 3D, stirred tank bioreactors, expanding hPSCs in suspension. Our data shows that hPSCs expand rapidly and efficiently in a 3L bioreactor, while retaining normal karyotyping and hPSC-associated characteristics. hPSCs are fed with an animal-free hPSC media, perfused into a closed, monitored and controlled, bioreactor, with no need for cell passaging. The platform supports expansion of hPSCs seeded in a bioreactor either when harvested from 2D cell culture or when thawed directly into the suspension vessel. Thawing cryopreserved cells directly into a suspension vessel eliminates the need for a 2D seed train and, therefore, greatly reduces labor, time and contributes to process aseptic, monitoring and automation. Furthermore, we show that 3D seed train from one suspension vessel to another is a feasible and viable solution for large-scale expansion of hPSCs. hPSCs expanded in a bioreactor are shown to have the capability to be directly differentiated in suspension. This expansion platform and associated attributes provides end-to-end, cryo-to-cryo solution for hPSC manufacturing. Expanding hPSCs in suspension, in a controlled bioreactor, results in high fold expansion without compromising cell quality, and the capacity of the cells to be further differentiated. This platform avoids 2D cell culture steps, reduces footprint, labor and cost, while enhancing process control and cell product quality.

Standardized qc assays and large scale expansion of pluripotent stem cells using an automated closed system.

Background & Aim Human pluripotent stem cells (hPSCs) hold great promise for disease modeling, drug discovery and clinical applications. Therefore, working with highly pluripotent, well characterized and quality controlled cell stocks during development is crucial to ensure reproducible experimental conditions. Methods, Results & Conclusion Here, we established a workflow encompassing stable expansion of hPSCs using a xeno-free cultivation medium, assessment of pluripotency using a defined marker combination and assessment of differentiation potential based on lineage-specific, complete media both combined with quantitative, multicolor flow cytometry analysis to characterize and quality control the cultivated hPSCs, as well as cryopreservation of hPSCs using an animal component-free, chemically defined cryopreservation media. Following this workflow, hPSCs could be stably expanded over 20 passages with persistent, high expression of pluripotency markers (>92%) TRA-1-60, SSEA-4, SSEA-5, Oct-4, Sox2 and almost no expression of differentiation maker SSEA-1, showed a homogenous morphology and also retained a stable karyotype. Quantitative, flow cytometry–based analysis reproducibly proved their differentiation potential into ectoderm, mesoderm, and endoderm. Cells cultivated under the same conditions showed a reproducible recovery after cryopreservation and thawing resulting in a 8-12 fold expansion in passage 1, high pluripotency marker expression as well as genomic stability confirmed 5 passages post thaw. Thus, the workflow shown here assures a standardized, robust expansion of hPSCs, includes characterization and quality control of the expanded cells, as well as efficient cryopreservation. The flow based QC strategy was also successfully applied for characterization of cells cultivated under standardized, automated, closed system conditions using the CliniMACS Prodigy Instrument which is of major relevance for generation of master cell banks (MCB) and working cell banks (WCB) for future clinical cell manufacturing.

Enhanced single-cell viability using 30Kc6 for efficient expansion of human induced pluripotent stem cells

Abstract Human pluripotent stem cells (hPSCs), such as human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs), have been widely used in stem cell research. hPSCs form colonies in culture dishes and are passaged as clumps. If the cells are dissociated into single cells, epithelial structures are disrupted, and rapid apoptosis is induced. The low viability of single cells restricts the passaging and expansion of hPSCs. Here, in order to enhance the single cell viability of hPSCs, we adopted an apoptotic strategy using the 30 Kc6 gene which inhibits mitochondrial apoptosis. With the expression of the 30 Kc6 gene in hiPSCs, no issues related to pluripotency or differentiation occurred. The hiPSC-30 Kc6 showed higher viability after the induction of apoptosis compared with control hiPSCs. Furthermore, hiPSC-30 Kc6 formed more colonies when the cells were enzymatically dissociated into single cells. Taken together, this study demonstrates that the 30 Kc6 gene could facilitate the passage and expansion of hiPSCs, potentially preventing the dissociation-induced apoptosis of single hPSCs.

Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement.

Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.

Improving single-cell cloning workflow for gene editing in human pluripotent stem cells

The availability of human pluripotent stem cells (hPSCs) and progress in genome engineering technology have altered the way we approach scientific research and drug development screens. Unfortunately, the procedures for genome editing of hPSCs often subject cells to harsh conditions that compromise viability: a major problem that is compounded by the innate challenge of single-cell culture. Here we describe a generally applicable workflow that supports single-cell cloning and expansion of hPSCs after genome editing and single-cell sorting. Stem-Flex and RevitaCell supplement, in combination with Geltrex or Vitronectin (VN), promote reliable single-cell growth in a feeder-free and defined environment. Characterization of final genome-edited clones reveals that pluripotency and normal karyotype are retained following this single-cell culture protocol. This time-efficient and simplified culture method paves the way for high-throughput hPSC culture and will be valuable for both basic research and clinical applications.

Meticulous optimization of cardiomyocyte yields in a 3-stage continuous integrated agitation bioprocess

Human pluripotent stem cells (hPSCs) can be a renewable source for generating cardiomyocyte (CM) for treating myocardial infraction. In our previous publication, we described an integrated microcarrier-based wave reactor process for the expansion and differentiation of hPSCs to CMs on a rocker based platform. However, this platform is limited in terms of linear scalability and CMs purity. The present study describes ways to overcome these limitations by the use of a stirred scalable platform and incorporation of an additional lactate based purification step which increases CM purity.Efficient CM differentiation in stirred spinners was achieved by (1) Addition of ascorbic acid (AS) during the differentiation phase which resulted in an increase of 38.42% in CM yield (0.84 ± 0.03 × 106vs 1.17 ± 0.07 × 106 CM/mL for cultures without AS and with AS respectively) and (2) Change of agitation regime to a shorter static intervals one (from 66 min off/6 min on (66/6) to 8 min off/1 min on (8/1)) during the first 3 days of differentiation which resulted in 22% increase in CM yield (1.50 ± 0.10 × 106vs 1.23 ± 0.07 × 106 CM/mL). The combination of AS addition and change in agitation regime resulted in a production yield of 1.50 ± 0.10 × 106 CM/mL which is comparable to that achieved in the rocker platform as described before (1.61 ± 0.36 × 106 CM/mL).Increase in CM purity was achieved by changing of culture medium to RPMI1640 (without glucose) + 5 mM lactate +0.6 mM AS at day 10 of differentiation which resulted in 44.5% increase in CM purity at day 15. The increase in purity of CMs was due to the death of the non-CM cells (~76% of cell death). It is important to note that in the absence of glucose, lactate was consumed at a rate of 0.01 mmol/106 cells/h. Addition of glucose, even in small amounts, during the purification step prevents the process of CM purification, due to the growth of the non-CM cell population.In summary, hPSC (hESC-HES3 and hiPSC-IMR90) can be efficiently differentiated to CMs in a scalable spinner process which integrates 7 days of expansion (3.01 ± 0.51 × 106 to 3.50 ± 0.65 × 106 cells/mL) followed by 10 days of WNT modulated CM differentiation and 5 days of lactate based purification. CM yield of 1.38 ± 0.22 × 106 to 1.29 ± 0.42 × 106 CM/mL with 72.5 ± 8.35% to 83.12 ± 8.73% cardiac troponin-T positive cells were obtained from these cultures.

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.