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This report summarizes the recent activity of the International Stem Cell Banking Initiative held at Harvard Stem Cell Institute, Boston, MA, USA, on June 18, 2017. In this meeting, we aimed to find consensus on ongoing issues of quality control (QC), safety, and efficacy of human pluripotent stem cell banks and their derivative cell therapy products for the global harmonization. In particular, assays for the QC testing such as pluripotency assays test and general QC testing criteria were intensively discussed. Moreover, the recent activities of global stem cell banking centers and the regulatory bodies were briefly summarized to provide an overview on global developments and issues. stem cells 2019;37:1130–1135, Photo of the 2017 Boston meeting, celebrating 10 years of International Stem Cell Banking Initiative activities.

Leukemia inhibitory factor (LIF) is widely used to establish and maintain naïve pluripotent stem cells, including mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Although the combination of chemical inhibitors called 2i can establish mouse iPSCs without LIF from primed pluripotent stem cells, it has been difficult, if not impossible, to establish mouse iPSCs from differentiated somatic cells without LIF. We previously showed that the fusion gene of the transactivation domain of MyoD and the full-length Oct4 (M3O) increases the efficiency of making iPSCs when transduced into fibroblasts along with Sox2, Klf4, and c-Myc (M3O-SKM). Here, we report that M3O-SKM allows for establishment of iPSCs without exogenous LIF from mouse embryonic fibroblasts. The established iPSCs remained undifferentiated and maintained pluripotency over 90days without LIF as long as M3O was expressed. The iPSCs upregulated miR-205-5p, which was potentially involved in the LIF-independence by suppressing the two signaling pathways inhibited by 2i. The result indicates that potentiated Oct4 can substitute for the LIF signaling pathway, providing a novel model to link Oct4 and LIF, two of the most significant players in naïve pluripotency.

Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

It has been very difficult, if not impossible, to establish mouse induced pluripotent stem cells (iPSCs) from differentiated cells, such as fibroblasts, without leukemia inhibitory factor (LIF). We have established and maintained LIF-independent iPSCs for longer than 120 days with modified Oct4 along with Sox2, Klf4, and c-Myc. The iPSCs will provide a novel tool to investigate the roles of the LIF-Stat3 signaling pathway in mouse pluripotent stem cells. Resource TableBiological reagent: induced pluripotent stem (iPS) cellName of Stem Cell constructM3O-lenti-iPSC-LIF(−)InstitutionUniversity of MinnesotaPerson who created resourceHiroyuki HiraiContact person and emailNobuaki Kikyo, kikyo001@umn.eduDate archived/stock date2014–2015OriginMouse embryonic fibroblastsType of resourceBiological reagent: induced pluripotent stem (iPS) cellSub-typeCell lineKey transcription factorsM3O, Sox2, Klf4, c-MycAuthenticationIdentity and purity of cell line confirmed (Figure 1)Link to related literature (direct URL links and full references)Hirai H., et al. In press, Stem Cell ResearchInformation in public databases

In this demonstration, spheroids formed from the β-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Numerous advances have been made in pancreatic β-cell replacement therapies for diabetes mellitus. While these therapies provide a positive impact and possible cure for the individual recipient, access is limited by availability of donor tissues. The derivation of pluripotent stem cells using efficient differentiation technologies has resulted in the generation of insulin-producing cells with characteristics similar to islet β-cells. Experimental transplantation studies have shown that these cells are capable of reducing hyperglycemia in short-term assays. Novel methodologies that facilitate the neogenesis of β-cells from endogenous hepatic or pancreatic tissue sources are also being investigated as a β-cell replacement strategy. Further research is necessary to protect these transplanted or regenerated cells from diabetic autoimmune pathology.

Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury-induced donor hepatocyte selection: the uPA/SCID mice and Fah(-/-) Rag2(-/-)Il2rg(-/-) mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah(-/-)Rag2(-/-) mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah(-/-)Rag2(-/-) mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines.

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5-6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.

To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.

Human embryonic stem cells, because of their unique combination of long-term self-renewal properties and pluripotency, are providing new avenues of investigation of stem cell biology and human development and show promise in providing a new source of human cells for transplantation therapies and pharmaceutical testing. Current methods of propagating these cells using combinations of mouse fibroblast feeder cultures and bovine serum components are inexpensive and, in general, useful. However, the systematic investigation of the regulation of self-renewal and the production of safer sources of cells for transplantation depends on the elimination of animal products and the use of defined culture conditions. Both goals are served by the development of serum-free culture methods for human embryonic stem cells.

In 2009 the International Stem Cell Banking Initiative (ISCBI) contributors and the Ethics Working Party of the International Stem Cell Forum published a consensus on principles of best practice for the procurement, cell banking, testing and distribution of human embryonic stem cell (hESC) lines for research purposes [1] , which was broadly also applicable to human induced pluripotent stem cell (hiPSC) lines. Here, we revisit this guidance to consider what the requirements would be for delivery of the early seed stocks of stem cell lines intended for clinical applications. The term 'seed stock' is used here to describe those cryopreserved stocks of cells established early in the passage history of a pluripotent stem cell line in the lab that derived the line or a stem cell bank, hereafter called the 'repository'. The seed stocks should provide cells with suitable documentation and provenance that would enable them to be taken forward for development in human therapeutic applications. WHO recommendations for the evaluation of animal cell cultures as substrates for the manufacture of biologicals and for the characterization of cell banks were updated in 2010 and provide a number of definitions and guiding principles that may apply to stem cells. The term 'cell bank' is used to describe a stock of vials or other containers of cells with consistent composition aliquoted from a single pool of cells of the same culture history (for other specific definitions see PAS 84 [2] and WHO [3] ).Three important assumptions have been made in the preparation of this document. First, that seed stocks of hPSCs are used as starting materials to make cell banks for use in clinical trials. The cell banks made within a clinical trial would need to be established according to Good Manufacturing Practice (GMP) in a facility with a relevant product manufacturing license. These banks would need additional risk assessment focused on the new banking process/reagents and the specific intended clinical application. Second, it has been assumed that undifferentiated pluripotent stem cells would not be inoculated into patients. Third, where feeder cells are used to culture hPSC lines, their cellular nature and intimate contact with the therapeutic cells means that they should be subject to similar risk assessment and banking procedures as applied to the hPSC cells.It is important to note that responsibility for establishing and updating national regulations for medicinal products relies on National Regulatory Authorities. Therefore, national requirements for cell therapy may vary considerably. Accordingly, it is not intended that this international consensus provides comprehensive guidance that will ensure compliance with requirements in any given jurisdiction. Rather, it is designed to aid the development of clinical grade materials by providing points to consider in the preparation of seed stocks of stem cell lines for use in cell therapy. It may arise that there are circumstances where it is not reasonably possible to meet specific procedures presented in this document. Where this is the case any alternative procedures should be justified and mitigate against any adverse consequences. Finally, this document could also serve as a useful reference to assist in the evaluation of potential sources of candidate cell lines for the development of cell-based medicines, and provide the links necessary to identify some of the key differences in regulatory requirements between countries.

Apoptosis-inducing factor (AIF) is an evolutionarily conserved, ubiquitously expressed flavoprotein with NADH oxidase activity that is normally confined to mitochondria. In mammalian cells, AIF is released from mitochondria in response to apoptotic stimuli and translocates to the nucleus where it is thought to bind DNA and contribute to chromatinolysis and cell death in a caspase-independent manner. Here we describe the consequences of inactivating Aif in the early mouse embryo. Unexpectedly, we found that both the apoptosis-dependent process of cavitation in embryoid bodies and apoptosis associated with embryonic neural tube closure occur in the absence of AIF, indicating that Aif function is not required for apoptotic cell death in early mouse embryos. By embryonic day 9 (E9), loss of Aif function causes abnormal cell death, presumably because of reduced mitochondrial respiratory chain complex I activity. Because of this cell death, Aif null embryos fail to increase significantly in size after E9. Remarkably, patterning processes continue on an essentially normal schedule, such that E10 Aif null embryos with only ≈1/10 the normal number of cells have the same somite number as their wild-type littermates. These observations show that pattern formation in the mouse can occur independent of embryo size and cell number.

BAF and PBAF are two related mammalian chromatin remodeling complexes essential for gene expression and development. PBAF, but not BAF, is able to potentiate transcriptional activation in vitro mediated by nuclear receptors, such as RXRalpha, VDR, and PPARgamma. Here we show that the ablation of PBAF-specific subunit BAF180 in mouse embryos results in severe hypoplastic ventricle development and trophoblast placental defects, similar to those found in mice lacking RXRalpha and PPARgamma. Embryonic aggregation analyses reveal that in contrast to PPARgamma-deficient mice, the heart defects are likely a direct result of BAF180 ablation, rather than an indirect consequence of trophoblast placental defects. We identified potential target genes for BAF180 in heart development, such as S100A13 as well as retinoic acid (RA)-induced targets RARbeta2 and CRABPII. Importantly, BAF180 is recruited to the promoter of these target genes and BAF180 deficiency affects the RA response for CRABPII and RARbeta2. These studies reveal unique functions of PBAF in cardiac chamber maturation.

The preservation of "stemness" in mouse embryonic stem (mES) cells is maintained through a signal transduction pathway that requires the gp130 receptor, the interleukin-6 (IL-6) family of cytokines, and the Janus Kinase-signal transducer and activator (JAK/STAT) pathway. The factors and signaling pathways that regulate "stemness" in human embryonic stem (hES) cells remain to be elucidated. Here we report that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feeder cells. Human ES cells differentiate in the presence of members of the IL-6 family of cytokines including leukemia inhibitory factor (LIF) and IL-6 or in the presence of the designer cytokine hyper-IL-6, which is a complex of soluble interleukin-6 receptor (IL-6R) and IL-6 with greatly enhanced bioactivity. Human ES cells express LIF, IL-6, and gp130 receptors, as well as the downstream signaling molecules. Stimulation of human and mouse ES cells with gp130 cytokines resulted in a robust phosphorylation of downstream ERK1, ERK2, and Akt kinases, as well as the STAT3 transcription factor. Loss of the pluripotency markers Nanog, Oct-4, and TRA-1-60 was observed in hES cells during gp130-dependent signaling, indicating that signaling through this pathway is insufficient to prevent the onset of differentiation. These data underscore a fundamental difference in requirements of murine versus hES cells. Furthermore, the data demonstrate the existence of an as-yet-unidentified factor in the conditioned media of mouse feeder layer cells that acts to maintain hES cell renewal in a STAT3-independent manner.

Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clinical uses have not yet been realized from the study of hES cells. Anticipation that these cells would be immediately useful for creating models of human disease has not yet been fulfilled. However, because of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for many areas of research and medicine. A major problem complicating developments in hES cell research is the difficulty of propagating and maintaining these cells in vitro without differentiation. This review addresses this problem and potential solutions in detail. In addition, the current state of research regarding the growth and maintenance of hES cells is summarized, along with basic protocols utilized by our laboratory for the successful propagation, characterization, and investigation of hES cells.

Human embryonic stem cells (hESCs) have the potential to differentiate to diverse cell types. This ability endows hESCs with promise for the development of novel therapeutics, as well as promise for the development of a rigorous genetic system to probe human gene function. However, in spite of the impending utility of hESCs for clinical and basic applications, little is known about their fundamental properties. Recent reports have documented transcriptional profiles of mouse embryonic stem cells (mESCs), adult stem cells and a single hESC line, H9. To date, however, the transcriptional profiles of independently-derived hESC lines have not been compared. In order to examine the similarities and differences in multiple hESC lines, we compared gene expression profiles of the HSF-1, HSF-6 and H9 lines. We found that the majority of genes examined were expressed in all three cell lines. However, we also observed that each line possessed a unique expression signature; the expression of many genes was limited to just one or two hESC lines. We suggest that the observed differences in gene expression between independently-derived hESC lines may reflect inherent differences in the initial culture of each line and/or the underlying genetics of the embryos from which the lines were derived.

Germ cells are the cells which ultimately give rise to mature sperm and eggs. In model organisms such as flies and worms, several genes that are required for formation and maintenance of germ cells have been identified and their interactions are rapidly being delineated. By contrast, little is known of the genes required for development of human germ cells and it is not clear whether findings from model organisms will translate into knowledge of human germ cell development, especially given observations that reproductive pathways may evolve more rapidly than somatic pathways. The Pumilio and Nanos genes have been especially well-characterized in model organisms and encode proteins that interact and are required for development of germ stem cells in one or both sexes. Here we report the first characterization of a mammalian Nanos homolog, human NANOS1 ( NOS1). We show that human NOS1 protein interacts with the human PUMILIO-2 (PUM2) protein via highly conserved domains to form a stable complex. We also show that in men, the NOS1 and PUM2 proteins are particularly abundant in germline stem cells. These observations mirror those in distant species and document for the first time a conserved protein-protein interaction in germ cells from flies to humans. These results suggest the possibility that the interaction of PUM2 and NOS1 may play a conserved role in germ cell development and maintenance in humans as in model organisms.

Early in development, a part of the embryo is set aside to become the germ cell lineage that will ultimately differentiate to form sperm and eggs and transmit genetic information to the next generation. Men with deletions encompassing the Y-chromosome DAZ genes have few or no germ cells but are otherwise healthy, indicating they harbor specific defects in formation or maintenance of germ cells. A DAZ homolog, DAZL ( DAZ-Like ), is found in diverse organisms, including humans and is required for germ cell development in males and/or females. We identified proteins that interact with DAZ proteins to better understand their function in human germ cells. Here, we show that PUM2, a human homolog of Pumilio, a protein required to maintain germ line stem cells in Drosophila and Caenorhabditis elegans , forms a stable complex with DAZ through the same functional domain required for RNA binding, protein–protein interactions and rescue of Pumilio mutations in flies. We also show that PUM2 is expressed predominantly in human embryonic stem cells and germ cells and colocalizes with DAZ and DAZL in germ cells. These data implicate PUM2 as a component of conserved cellular machinery that may be required for germ cell development.

A major limitation of the use of cellular therapies is the loss of donor-derived cells because of immune incompatibility. While induced pluripotent stem (iPS) cells offer the potential for autologous transplant therapies, questions have been raised using a mouse model that specific antigens mediate the rejection of grafts after syngeneic transplants with iPS, but not embryonic stem (ES) cells. In this study, we examined whether the human homologs of these markers, HORMAD1, ZG16, and Cyp3A, are differentially expressed in human iPS versus ES cells, as well as undifferentiated and in vitro-differentiated cells. Both qRT-PCR and flow cytometric analyses demonstrated similar gene and protein expression profiles for iPS and ES cells regardless of differentiation state. Our data are consistent with a recent study in mice that showed no evidence of rejection of differentiated syngeneic iPS cells. Furthermore, our results suggest that expression of these gene products cannot predict differences in clinical outcomes between human iPS and ES-derived cells.

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present inPbx1−/−fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1−/−embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted inPbx1−/−embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated.

Abstract —Transforming growth factor-β 1 (TGF-β 1 ) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-β 1 expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-β 1 in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-β 1 is expressed from the SM22α promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22α-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22α–β-galactosidase transgene. SM22α– β-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22α–TGF-β 1 transgenic mice died before E11.5. SM22α–TGF-β 1 transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-β 1 from the SM22α promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-β 1 expression in adults may require a conditional transgenic approach. Moreover, because the SM22α promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22α promoter to drive expression of “vasculoactive” transgenes may be particularly likely to cause embryonic death.

Cellular therapies are emerging as a standard approach for the treatment of several diseases. However, realizing the promise of cellular therapies across the full range of treatable disorders will require large-scale, controlled, reproducible culture methods. Bioreactor systems offer the scale-up and monitoring needed, but standard stirred bioreactor cultures do not allow for the real-time regulation of key nutrients in the medium. In this study, β-TC6 insulinoma cells were aggregated and cultured for 3 weeks as a model of manufacturing a mammalian cell product. Cell expansion rates and medium nutrient levels were compared in static, stirred suspension bioreactors (SSB), and continuously fed (CF) SSB. While SSB cultures facilitated increased culture volumes, no increase in cell yields were observed, partly due to limitations in key nutrients, which were consumed by the cultures between feedings, such as glucose. Even when glucose levels were increased to prevent depletion between feedings, dramatic fluctuations in glucose levels were observed. Continuous feeding eliminated fluctuations and improved cell expansion when compared with both static and SSB culture methods. Further improvements in growth rates were observed after adjusting the feed rate based on calculated nutrient depletion, which maintained physiological glucose levels for the duration of the expansion. Adjusting the feed rate in a continuous medium replacement system can maintain the consistent nutrient levels required for the large-scale application of many cell products. Continuously fed bioreactor systems combined with nutrient regulation can be used to improve the yield and reproducibility of mammalian cells for biological products and cellular therapies and will facilitate the translation of cell culture from the research lab to clinical applications.

A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.

Cytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (delta- cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated delta-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 x 10(3) delta-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor- derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10-20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development. ispartof: PLOS ONE vol:5 issue:8 ispartof: location:United States status: published

Human embryonic stem cells (hESCs) have the potential to differentiate into all cell types in the body and hold great promise for regenerative medicine; however, large-scale expansion of undifferentiated hESCs remains a major challenge. Self-renewal of hESCs requires culturing these cells on either mouse or human fibroblast cells (i.e., a feeder layer of cells), or on artificial extracellular matrices (ECMs) while supplementing the media with soluble growth factors. Here we report a completely synthetic ECM system composed of a semi-interpenetrating polymer network (sIPN), a polymer hydrogel, which was designed to allow the independent manipulation of cell adhesion ligand presentation and matrix stiffness. In the short term, hESCs that were cultured on the sIPN adhered to the surface, remained viable, maintained the morphology, and expressed the markers of undifferentiated hESCs. This was the first demonstration that a completely synthetic ECM can support short-term self-renewal of hESCs.

Transforming growth factor beta (TGFbeta) plays an important role in development and maintenance of murine yolk sac vascular development. Targeted deletions of Tgfb1 and other components of this signaling pathway, such as Acvrl1, Tgfbr1 and Tgfbr2, result in abnormal vascular development especially of the yolk sac, leading to embryonic lethality. There are significant differences between murine and primate development that limit interpretation of studies from mouse models. Thus, to examine the role of TGFbeta in early human vascular development we used the model of differentiating human embryonic stem cell-derived embryoid bodies to recapitulate early stages of embryonic development. TGFbeta was applied for different time frames after initiation of embryoid body cultures to assess its effect on differentiation. TGFbeta inhibited the expression of endodermal, endothelial and hematopoietic markers, which contrasts with findings in the mouse in which TGFbeta reduced the level of endodermal markers but increased endothelial marker expression. The inhibition observed was not due to changes in proliferation or apoptosis. This marked contrast between the two species may reflect the different origins of the yolk sac hemangiogenic lineages in mouse and human. TGFbeta effects on the hypoblast, from which these cell lineages are derived in human, would decrease subsequent differentiation of hematopoietic, endothelial and endodermal cells. By contrast, TGFbeta action on murine hypoblast, while affecting endoderm would not affect the hemangiogenic lineages that are epiblast-derived in the mouse. This study highlights important differences between early human and mouse embryonic development and suggests a role of TGFbeta in human hypoblast differentiation., Journal Article, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain "stemness" in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self-renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct-4, nanog, and TRA-1-60 and remained pluripotent, as shown by the in vivo formation of teratomas.

Genes required to maintain pluripotency in human embryonic stem (hES) cells are largely unknown, with the exception of OCT-4, a homolog of mouse Oct-4, which is critical for the establishment of the embryonic inner cell mass and the generation of totipotent mouse embryonic stem (mES) cell lines. In the current study, we identified two genes with expression similar to OCT-4, in that they are largely restricted to pluripotent hES cells, premeiotic germ lineage cells, and testicular germ cell tumor cells. Furthermore, we determined that upon hES cell differentiation, their expression is downregulated. The genes we identified in the current study include the human stella-related (STELLAR) gene, which encodes a highly divergent protein (with just 32.1% identity to mouse stella over the 159 amino acid sequence) that maps to human chromosome 12p13. Notably, human STELLAR is located distal to a previously uncharacterized homeobox gene, which is the human homolog of the recently identified murine gene, Nanog, and proximal to the GDF3 locus, whose transcription is restricted to germ cell tumor cells. Our characterization of STELLAR, NANOG, and GDF3 suggests that they may play a similar role in humans as in mice, in spite of their remarkable evolutionary divergence.

Little is known of molecular requirements for specification of human germ cells. However, it is likely that they are specified through the action of sequentially expressed genes just as in model organisms. We sought to determine whether human embryonic stem (ES) cell lines, like those of mice, might be capable of forming germ cells in vitro. We compared transcriptional profiles of three pluripotent human ES cells to those of isolated inner cell mass (ICM) cells. We found that ICM cells expressed NANOS1, STELLAR and OCT4, whereas undifferentiated human ES cells expressed these genes along with the germ cell-specific gene, DAZL. Upon ES cell differentiation into embryoid bodies (EBs), we observed a shift in expression from RNA and protein markers of immature germ cells to those indicative of mature germ cells, including expression of VASA, BOL, SCP1, SCP3, GDF9 and TEKT1. Although ability to test the function of these putative VASA positive germ cells is limited, these results demonstrate that differentiation of human ES cells into EBs in vitro results in formation of cells that express markers specific to gonocytes.

The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells, Furthermore, higher percentages of CD33(+)CD15(-) cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies. (C) 1999 International Society for Experimental Hematology, Published by Elsevier Science Inc.

Highly purified CD34++CD38−Lin− hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli β-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the β-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38−Lin− cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.

The generation of blood cells, haematopoiesis, in the mouse embryo begins with the development of primitive nucleated erythroid cells in the yolk sac followed by the appearance of precursors for multiple definitive haematopoietic lineages1–4. The later developing lineages arise from multipotential stem cells5,6, but the relationship of primitive erythroid cells to these other haematopoietic populations is unknown. Using an in vitro embryonic stem (ES) cell differentiation system7, we show that primitive erythrocytes and other haematopoietic lineages arise from a common multipotential precursor that develops within embryoid bodies generated from differentiated ES cells. In response to vascular endothelial growth factor and c-kit ligand these precursors give rise to colonies containing immature cells (blasts) expressing marker genes characteristic of haematopoietic precursors. Many blast colonies also expressed βH1 and β major globins but not Brachyury, a mesodermal marker. Kinetic analysis demonstrated that the blast colony-forming cells represent a transient population, preceding the establishment of the primitive erythroid and other lineage-restricted precursors. This precursor population may represent the earliest stage of embryonic haematopoietic commitment.