Non-xenogeneic expansion and definitive endoderm differentiation of human pluripotent stem cells in an automated bioreactor.

Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic and induced pluripotent stem cells to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with the maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.

MicroRNA-7 directly targets Reg1 in pancreatic cells.

Regenerating islet-derived (Reg) proteins, which were first discovered in the pancreas, are associated with increased proliferation, prevention of apoptosis, and enhanced differentiation in normal and disease states, but very little is known about the regulation of their expression. We hypothesized that Reg expression is influenced by microRNAs. Bioinformatic analysis predicted Reg1 to be a target of microRNA-7 (miR-7), which influences pancreatic ß-cell function. To this end, we investigated the effects of miR-7 on Reg1 expression in pancreatic acinar and islet ß-cells. High levels of Reg1 were noted by immunostaining and Western blotting in acinar cells in contrast to islet cells. A reciprocal expression pattern was observed for miR-7. Overexpression of miR-7 resulted in Reg1 mRNA suppression and reduction of secreted Reg1 protein. Conversely, miR-7 knockdown led to increases in Reg1. Targeting of Reg1 by miR-7 was confirmed via luciferase activity assays. In contrast, miR-7 did not directly repress the human ortholog of Reg1 REG1A as well as REG1B indicating species differences in the regulation of Reg expression. This is the first account of microRNA modulation of any Reg member warranting studies to fill gaps in our knowledge of Reg protein biology, particularly in disease contexts.

Who Will Win: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells for ß Cell Replacement and Diabetes Disease Modeling?

PURPOSE OF REVIEW: Ever since the reprogramming of human fibroblasts to induced pluripotent stem cells (hiPSCs), scientists have been trying to determine if hiPSCs can give rise to progeny akin to native terminally differentiated cells as human embryonic stem cells (hESCs) do. Many different somatic cell types have been successfully reprogrammed via a variety of methods. In this review, we will discuss recent studies comparing hiPSCs and hESCs and their ability to differentiate to desired cell types as well as explore diabetes disease models. RECENT FINDINGS: Both somatic cell origin and the reprogramming method are important to the epigenetic state of the hiPSCs; however, genetic background contributes the most to differences seen between hiPSCs and hESCs. Based on our review of the relevant literature, hiPSCs display differences compared to hESCs, including a higher propensity for specification toward particular cell types based on memory retained from the somatic cell of origin. Moreover, hiPSCs provide a unique opportunity for creating diabetes disease models.

Distinct allelic patterns of nanog expression impart embryonic stem cell population heterogeneity.

Nanog is a principal pluripotency regulator exhibiting a disperse distribution within stem cell populations in vivo and in vitro. Increasing evidence points to a functional role of Nanog heterogeneity on stem cell fate decisions. Allelic control of Nanog gene expression was reported recently in mouse embryonic stem cells. To better understand how this mode of regulation influences the observed heterogeneity of NANOG in stem cell populations, we assembled a multiscale stochastic population balance equation framework. In addition to allelic control, gene expression noise and random partitioning at cell division were considered. As a result of allelic Nanog expression, the distribution of Nanog exhibited three distinct states but when combined with transcriptional noise the profile became bimodal. Regardless of their allelic expression pattern, initially uniform populations of stem cells gave rise to the same Nanog heterogeneity within ten cell cycles. Depletion of NANOG content in cells switching off both gene alleles was slower than the accumulation of intracellular NANOG after cells turned on at least one of their Nanog gene copies pointing to Nanog state-dependent dynamics. Allelic transcription of Nanog also raises issues regarding the use of stem cell lines with reporter genes knocked in a single allelic locus. Indeed, significant divergence was observed in the reporter and native protein profiles depending on the difference in their half-lives and insertion of the reporter gene in one or both alleles. In stem cell populations with restricted Nanog expression, allelic regulation facilitates the maintenance of fractions of self-renewing cells with sufficient Nanog content to prevent aberrant loss of pluripotency. Our findings underline the role of allelic control of Nanog expression as a prime determinant of stem cell population heterogeneity and warrant further investigation in the contexts of stem cell specification and cell reprogramming.

Light-Mediated Enhancement of Glucose-Stimulated Insulin Release of Optogenetically Engineered Human Pancreatic Beta-Cells.

Enhancement of glucose-stimulated insulin secretion (GSIS) in exogenously delivered pancreatic ß-cells is desirable, for example, to overcome the insulin resistance manifested in type 2 diabetes or to reduce the number of ß-cells for supporting homeostasis of blood sugar in type 1 diabetes. Optogenetically engineered cells can potentiate their function with exposure to light. Given that cyclic adenosine monophosphate (cAMP) mediates GSIS, we surmised that optoamplification of GSIS is feasible in human ß-cells carrying a photoactivatable adenylyl cyclase (PAC). To this end, human EndoC-ßH3 cells were engineered to express a blue-light-activated PAC, and a workflow was established combining the scalable manufacturing of pseudoislets (PIs) with efficient adenoviral transduction, resulting in over 80% of cells carrying PAC. Changes in intracellular cAMP and GSIS were determined with the photoactivation of PAC in vitro as well as after encapsulation and implantation in mice with streptozotocin-induced diabetes. cAMP rapidly rose in ß-cells expressing PAC with illumination and quickly declined upon its termination. Light-induced amplification in cAMP was concomitant with a greater than 2-fold GSIS vs ß-cells without PAC in elevated glucose. The enhanced GSIS retained its biphasic pattern, and the rate of oxygen consumption remained unchanged. Diabetic mice receiving the engineered ß-cell PIs exhibited improved glucose tolerance upon illumination compared to those kept in the dark or not receiving cells. The findings support the use of optogenetics for molecular customization of the ß-cells toward better treatments for diabetes without the adverse effects of pharmacological approaches.

An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function.

Bioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life-altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here is report an integrated optogenetic and bioelectronic platform for stable and long-term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose-dependent and time-limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi-electrode array that measures real-time electrophysiological responses at 32 spatially-distinct locations. Irradiation at 27 µW mm-2 results in a 14% elevation of the beating rate within 20-25 min, which remains stable for at least 2 h. The beating rate can be cycled through "on" and "off" light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed-loop systems for cardiac regulation and intervention, for example, in the context of arrythmias.

Mathematical modeling clarifies the paracrine roles of insulin and glucagon on the glucose-stimulated hormonal secretion of pancreatic alpha- and beta-cells.

Introduction: Blood sugar homeostasis relies largely on the action of pancreatic islet hormones, particularly insulin and glucagon. In a prototypical fashion, glucagon is released upon hypoglycemia to elevate glucose by acting on the liver while elevated glucose induces the secretion of insulin which leads to sugar uptake by peripheral tissues. This simplified view of glucagon and insulin does not consider the paracrine roles of the two hormones modulating the response to glucose of α- and ß-cells. In particular, glucose-stimulated glucagon secretion by isolated α-cells exhibits a Hill-function pattern, while experiments with intact pancreatic islets suggest a 'U'-shaped response. Methods: To this end, a framework was developed based on first principles and coupled to experimental studies capturing the glucose-induced response of pancreatic α- and ß-cells influenced by the two hormones. The model predicts both the transient and steady-state profiles of secreted insulin and glucagon, including the typical biphasic response of normal ß-cells to hyperglycemia. Results and discussion: The results underscore insulin activity as a differentiating factor of the glucagon secretion from whole islets vs. isolated α-cells, and highlight the importance of experimental conditions in interpreting the behavior of islet cells in vitro. The model also reproduces the hyperglucagonemia, which is experienced by diabetes patients, and it is linked to a failure of insulin to inhibit α-cell activity. The framework described here is amenable to the inclusion of additional islet cell types and extrapancreatic tissue cells simulating multi-organ systems. The study expands our understanding of the interplay of insulin and glucagon for pancreas function in normal and pathological conditions.

An Integrated Optogenetic and Bioelectronic Platform for Regulating Cardiomyocyte Function.

We report an integrated optogenetic and bioelectronic platform for stable and long-term modulation and monitoring of cardiomyocyte function in vitro. Optogenetic inputs were achieved through expression of a photoactivatable adenylyl cyclase (bPAC), that when activated by blue light caused a dose-dependent and time-limited increase in autonomous cardiomyocyte beat rate. Bioelectronic readouts were achieved through an integrated planar multi-electrode array (MEA) that provided real-time readouts of electrophysiological activity from 32 spatially-distinct locations. Irradiation at 27 µW/mm2 resulted in a ca. 14% increase in beat rate within 20-25 minutes, which remained stable for at least 2 hours. The beating rate could be cycled through repeated "on" and "off' states, and its magnitude was a monotonic function of irradiation intensity. Our integrated platform opens new avenues in bioelectronic medicine, including closed-loop feedback systems, with potential applications for cardiac regulation including arrhythmia diagnosis and intervention.

A novel nuclear FGF Receptor-1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells.

FGF Receptor-1 (FGFR1), a membrane-targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal-like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA-induced differentiation while full-length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co-associate with 5'-Fluorouridine-labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf-2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter-gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation.

Scaling down the size and increasing the throughput of glycosyltransferase assays: activity changes on stem cell differentiation.

Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall ß(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galß1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability.

Emerging molecular technologies for light-mediated modulation of pancreatic beta-cell function.

BACKGROUND: Optogenetic modalities as well as optochemical and photopharmacological strategies, collectively termed optical methods, have revolutionized the control of cellular functions via light with great spatiotemporal precision. In comparison to the major advances in the photomodulation of signaling activities noted in neuroscience, similar applications to endocrine cells of the pancreas, particularly insulin-producing ß-cells, have been limited. The availability of tools allowing light-mediated changes in the trafficking of ions such as K+ and Ca2+ and signaling intermediates such as cyclic adenosine monophosphate (cAMP), renders ß-cells and their glucose-stimulated insulin secretion (GSIS) amenable to optoengineering for drug-free control of blood sugar. SCOPE OF REVIEW: The molecular circuit of the GSIS in ß-cells is described with emphasis on intermediates which are targetable for optical intervention. Various pharmacological agents modifying the release of insulin are reviewed along with their documented side effects. These are contrasted with optical approaches, which have already been employed for engineering ß-cell function or are considered for future such applications. Principal obstacles are also discussed as the implementation of optogenetics is pondered for tissue engineering and biology applications of the pancreas. MAJOR CONCLUSIONS: Notable advances in optogenetic, optochemical and photopharmacological tools are rendering feasible the smart engineering of pancreatic cells and tissues with light-regulated function paving the way for novel solutions for addressing pancreatic pathologies including diabetes.

Proteomic Analysis of Exosomes during Cardiogenic Differentiation of Human Pluripotent Stem Cells.

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases.

Emerging routes to the generation of functional ß-cells for diabetes mellitus cell therapy.

Diabetes mellitus, which affects more than 463 million people globally, is caused by the autoimmune ablation or functional loss of insulin-producing ß-cells, and prevalence is projected to continue rising over the next decades. Generating ß-cells to mitigate the aberrant glucose homeostasis manifested in the disease has remained elusive. Substantial advances have been made in producing mature ß-cells from human pluripotent stem cells that respond appropriately to dynamic changes in glucose concentrations in vitro and rapidly function in vivo following transplantation in mice. Other potential avenues to produce functional ß-cells include: transdifferentiation of closely related cell types (for example, other pancreatic islet cells such as α-cells, or other cells derived from endoderm); the engineering of non-ß-cells that are capable of modulating blood sugar; and the construction of synthetic 'cells' or particles mimicking functional aspects of ß-cells. This Review focuses on the current status of generating ß-cells via these diverse routes, highlighting the unique advantages and challenges of each approach. Given the remarkable progress in this field, scalable bioengineering processes are also discussed for the realization of the therapeutic potential of derived ß-cells.

Xenogeneic-Free System for Biomanufacturing of Cardiomyocyte Progeny From Human Pluripotent Stem Cells.

Functional heart cells and tissues sourced from human pluripotent stem cells (hPSCs) have great potential for substantially advancing treatments of cardiovascular maladies. Realization of this potential will require the development of cost-effective and tunable bioprocesses for manufacturing hPSC-based cell therapeutics. Here, we report the development of a xeno-free platform for guiding the cardiogenic commitment of hPSCs. The system is based on a fully defined, open-source formulation without complex supplements, which have varied and often undetermined effects on stem cell physiology. The formulation was used to systematically investigate factors inducing the efficient commitment to cardiac mesoderm of three hPSC lines. Contractile clusters of cells appeared within a week of differentiation in planar cultures and by day 13 over 80% of the cells expressed cardiac progeny markers such as TNNT2. In conjunction with expansion, this differentiation strategy was employed in stirred-suspension cultures of hPSCs. Scalable differentiation resulted in 0.4-2 million CMs/ml or ∼5-20 TNNT2-positive cells per seeded hPSC without further enrichment. Our findings will contribute to the engineering of bioprocesses advancing the manufacturing of stem cell-based therapeutics for heart diseases.

Amelioration of Diabetes in a Murine Model upon Transplantation of Pancreatic ß-Cells with Optogenetic Control of Cyclic Adenosine Monophosphate.

Pharmacological augmentation of glucose-stimulated insulin secretion (GSIS), for example, to overcome insulin resistance in type 2 diabetes is linked to suboptimal regulation of blood sugar. Cultured ß-cells and islets expressing a photoactivatable adenylyl cyclase (PAC) are amenable to GSIS potentiation with light. However, whether PAC-mediated enhancement of GSIS can improve the diabetic state remains unknown. To this end, ß-cells were engineered with stable PAC expression that led to over 2-fold greater GSIS upon exposure to blue light while there were no changes in the absence of glucose. Moreover, the rate of oxygen consumption was unaltered despite the photoinduced elevation of GSIS. Transplantation of these cells into streptozotocin-treated mice resulted in improved glucose tolerance, lower hyperglycemia, and higher plasma insulin when subjected to illumination. Embedding optogenetic networks in ß-cells for physiologically relevant control of GSIS will enable novel solutions potentially overcoming the shortcomings of current treatments for diabetes.

Four Decades After the Discovery of Regenerating Islet-Derived (Reg) Proteins: Current Understanding and Challenges.

Regenerating islet-derived (Reg) proteins have emerged as multifunctional agents with pro-proliferative, anti-apoptotic, differentiation-inducing and bactericidal properties. Over the last 40 years since first discovered, Reg proteins have been implicated in a gamut of maladies including diabetes, various types of cancer of the digestive tract, and Alzheimer disease. Surprisingly though, a consensus is still absent on the regulation of their expression, and molecular underpinning of their function. Here, we provide a critical appraisal of recent findings in the field of Reg protein biology. Specifically, the structural characteristics are reviewed particularly in connection with established or purported functions of different members of the Reg family. Moreover, Reg expression patterns in different tissues both under normal and pathophysiological conditions are summarized. Putative receptors and cascades reported to relay Reg signaling inciting cellular responses are presented aiming at a better appreciation of the biological activities of the distinct Reg moieties. Challenges are also discussed that have hampered thus far the rapid progress in this field such as the use of non-standard nomenclature for Reg molecules among various research groups, the existence of multiple Reg members with significant degree of homology and possibly compensatory modes of action, and the need for common assays with robust readouts of Reg activity. Coordinated research is warranted going forward, given that several research groups have independently linked Reg proteins to diseased states and raised the possibility that these biomolecules can serve as therapeutic targets and biomarkers.

Stem/Progenitor cell sources of insulin-producing cells for the treatment of diabetes.

Patients with diabetes experience decreased insulin secretion that is linked to a significant reduction in the number of islet cells. Reversal of diabetes can be achieved through islet transplantation, but the scarcity of donor islets severely hinders wide application of this therapeutic modality. Toward that end, embryonic stem cells, adult tissue-residing progenitor cells, and regenerating native beta-cells may serve as sources of islet cell surrogates. Insulin-producing cells generated from stem or progenitor cells display subsets of native beta-cell attributes, indicating the need for further development of methods for differentiation to completely functional beta-cells. Pharmacological approaches aiming at stimulating the in vivo/ex vivo regeneration of beta-cells have also been proposed as a way of augmenting islet cell mass. We review the current state of the generation of insulin-producing cells from different sources with emphasis on embryonic stem cells and adult progenitor cells. Challenges for the clinical use of these sources are also discussed.

Optogenetic regulation of insulin secretion in pancreatic ß-cells.

Pancreatic ß-cell insulin production is orchestrated by a complex circuitry involving intracellular elements including cyclic AMP (cAMP). Tackling aberrations in glucose-stimulated insulin release such as in diabetes with pharmacological agents, which boost the secretory capacity of ß-cells, is linked to adverse side effects. We hypothesized that a photoactivatable adenylyl cyclase (PAC) can be employed to modulate cAMP in ß-cells with light thereby enhancing insulin secretion. To that end, the PAC gene from Beggiatoa (bPAC) was delivered to ß-cells. A cAMP increase was noted within 5 minutes of photostimulation and a significant drop at 12 minutes post-illumination. The concomitant augmented insulin secretion was comparable to that from ß-cells treated with secretagogues. Greater insulin release was also observed over repeated cycles of photoinduction without adverse effects on viability and proliferation. Furthermore, the expression and activation of bPAC increased cAMP and insulin secretion in murine islets and in ß-cell pseudoislets, which displayed a more pronounced light-triggered hormone secretion compared to that of ß-cell monolayers. Calcium channel blocking curtailed the enhanced insulin response due to bPAC activity. This optogenetic system with modulation of cAMP and insulin release can be employed for the study of ß-cell function and for enabling new therapeutic modalities for diabetes.

Human pluripotent stem cell differentiation to functional pancreatic cells for diabetes therapies: Innovations, challenges and future directions.

Recent advances in the expansion and directed pancreatogenic differentiation of human pluripotent stem cells (hPSCs) have intensified efforts to generate functional pancreatic islet cells, especially insulin-secreting ß-cells, for cell therapies against diabetes. However, the consistent generation of glucose-responsive insulin-releasing cells remains challenging. In this article, we first present basic concepts of pancreatic organogenesis, which frequently serves as a basis for engineering differentiation regimens. Next, past and current efforts are critically discussed for the conversion of hPSCs along pancreatic cell lineages, including endocrine ß-cells and α-cells, as well as exocrine cells with emphasis placed on the later stages of commitment. Finally, major challenges and future directions are examined, such as the identification of factors for in vivo maturation, large-scale culture and post processing systems, cell loss during differentiation, culture economics, efficiency, and efficacy and exosomes and miRNAs in pancreatic differentiation.

Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing.

The development of platforms for the expansion and directed differentiation of human pluripotent stem cells (hPSCs) in large quantities under xeno-free conditions is a key step toward the realization of envisioned stem cell-based therapies. Microcarrier bioreactors afford great surface-to-volume ratio, scalability and customization with typical densities of 106-107 cells/ml or higher. In this study, a simple and inexpensive method was established for generating microcarriers without animal-derived components. While coating polystyrene beads with vitronectin alone did not support the culture of hPSCs in stirred suspension, the inclusion of recombinant human serum albumin and UV irradiation led to enhanced seeding efficiency and retention while cells grew more than 20-fold per passage for multiple successive passages and without loss of cell pluripotency. Human PSCs expanded on microcarriers were coaxed to tri-lineage differentiation demonstrating that this system can be used for the self-renewal and specification of hPSCs to therapeutically relevant cell types. Such systems will be critical for the envisioned use of stem cells in regenerative medicine and drug discovery.