Suspended in culture--human pluripotent cells for scalable technologies

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively termed human pluripotent stem cells (hPSCs), are typically derived and maintained in adherent and semi-defined culture conditions. Recently a number of groups, including Chen et al., 2012, have demonstrated that hESCs can now be expanded efficiently and maintain pluripotency over long-term passaging as aggregates in a serum-free defined suspension culture system, permitting the preparation of scalable cGMP derived hPSC cultures for cell banking, high throughput research programs and clinical applications. In this short commentary we describe the utility and potential future uses of suspension culture systems for hPSCs.     

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.     

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.     

ROCK Inhibitor Is Not Required for Embryoid Body Formation from Singularized Human Embryonic Stem Cells

We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.     

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment

Background  

We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.     

Primary bone-derived cells induce osteogenic differentiation without exogenous factors in human embryonic stem cells

We developed a new and efficient method for osteoblastic differentiation of human embryonic stem cells (hESCs) using primary bone-derived cells (PBDs). Three days after embryoid body (hEB) formation, cells were allowed to adhere to culture surface where PBDs were pre-plated and mitomycin C-treated in DMEM/F12 medium supplemented with 5% knockout serum replacement. As early as 14 days, mineralization and formation of nodule-like structures in cocultured hEBs were prominent by von Kossa and Alizarin S staining, and expressions of osteoblast-specific markers including bone sialoprotein, alkaline phosphates, osteocalcin, collagen 1, and core binding factor alpha1 by RT-PCR. In addition, FACS analysis revealed that over 19% of the differentiated cells expressed osteocalcin. These results suggest that PBDs not only have osteogenic effects releasing osteogenic factors as bone morphogenic protein (BMP) 2 and BMP 4 but also have exerted other effects, whether chemical or physical, for the differentiation of hESCs.     

Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.     

One-step derivation of cardiomyocytes and mesenchymal stem cells from human pluripotent stem cells

Cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) are important cell types for cardiac repair post myocardial infarction. Here we proved that both CMs and MSCs can be simultaneously generated from human induced pluripotent stem cells (hiPSCs) via a pro-mesoderm differentiation strategy. Two hiPSC lines, hiPSC (1) and hiPSC (2) were generated from human dermal fibroblasts using OCT-4, SOX-2, KLF-4, c-Myc via retroviral-based reprogramming. H9 human embryonic stem cells (hESCs) served as control. CMs and MSCs were co-generated from hiPSCs and hESCs via embryoid body-dependent cardiac differentiation protocol involving a serum-free and insulin-depleted medium containing a p38 MAPK inhibitor, SB 203580. Comparing to bone marrow and umbilical cord blood-derived MSCs, hiPSC-derived MSCs (iMSCs) expressed common MSC markers and were capable of adipogenesis, osteogenesis and chondrogenesis. Moreover, iMSCs continuously proliferated for more than 32 population doublings without cellular senescence and showed superior pro-angiogenic and wound healing properties. In summary, we generated a large number of homogenous MSCs in conjunction with CMs in a low-cost and efficient one step manner. Functionally competent CMs and MSCs co-generated from hiPSCs may be useful for autologous cardiac repair.     

DNA methylation dynamics in human induced pluripotent stem cells

Indeed human induced pluripotent stem cells (hiPSCs) are considered to be powerful tools in regenerative medicine. To enable the use of hiPSCs in the field of regenerative medicine, it is necessary to understand the mechanisms of reprogramming during the transformation of somatic cells into hiPSCs. Genome-wide epigenetic modification constitutes a critical event in the generation of iPSCs. In other words, to analyze epigenetic changes in iPSCs means to elucidate reprogramming processes. We have established a large number of hiPSCs derived from various human tissues and have obtained their DNA methylation profiles. Comparison analyses indicated that the epigenetic patterns of various hiPSCs, irrespective of their source tissue, were very similar to one another and were similar to those of human embryonic stem cells (hESCs). However, the profiles of hiPSCs and hESCs exhibited epigenetic differences, which were caused by random aberrant hypermethylation at early passages. Interestingly, continuous passaging of the hiPSCs diminished the differences between DNA methylation profiles of hiPSCs and hESCs. The number of aberrant DNA methylation regions may thus represent a useful epigenetic index for evaluating hiPSCs in the context of therapeutic applications.     

The HOXB4 homeoprotein promotes the ex vivo enrichment of functional human embryonic stem cell-derived NK cells

Human embryonic stem cells (hESCs) can be induced to differentiate into blood cells using either co-culture with stromal cells or following human embryoid bodies (hEBs) formation. It is now well established that the HOXB4 homeoprotein promotes the expansion of human adult hematopoietic stem cells (HSCs) but also myeloid and lymphoid progenitors. However, the role of HOXB4 in the development of hematopoietic cells from hESCs and particularly in the generation of hESC-derived NK-progenitor cells remains elusive. Based on the ability of HOXB4 to passively enter hematopoietic cells in a system that comprises a co-culture with the MS-5/SP-HOXB4 stromal cells, we provide evidence that HOXB4 delivery promotes the enrichment of hEB-derived precursors that could differentiate into fully mature and functional NK. These hEB-derived NK cells enriched by HOXB4 were characterized according to their CMH class I receptor expression, their cytotoxic arsenal, their expression of IFNγ and CD107a after stimulation and their lytic activity. Furthermore our study provides new insights into the gene expression profile of hEB-derived cells exposed to HOXB4 and shows the emergence of CD34(+)CD45RA(+) precursors from hEBs indicating the lymphoid specification of hESC-derived hematopoietic precursors. Altogether, our results outline the effects of HOXB4 in combination with stromal cells in the development of NK cells from hESCs and suggest the potential use of HOXB4 protein for NK-cell enrichment from pluripotent stem cells.     

Scalable expansion of human pluripotent stem cells in suspension culture

Routine commercial and clinical applications of human pluripotent stem cells (hPSCs) and their progenies will require increasing cell quantities that cannot be provided by conventional adherent culture technologies. Here we describe a straightforward culture protocol for the expansion of undifferentiated human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) in suspension culture. This culture technique was successfully tested on two hiPSC clones, three hESC lines and on a nonhuman primate ESC line. It is based on a defined medium and single-cell inoculation, but it does not require culture preadaptation, use of microcarriers or any other matrices. Over a time course of 4-7 d, hPSCs can be expanded up to sixfold. Preparation of a high-density culture and its subsequent translation to scalable stirred suspension in Erlenmeyer flasks and stirred spinner flasks are also feasible. Importantly, hPSCs maintain pluripotency and karyotype stability for more than ten passages.     

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.