Hypoxia within subcutaneously implanted macroencapsulation devices limits the viability and functionality of densely loaded islets.

Introduction: Subcutaneous macroencapsulation devices circumvent disadvantages of intraportal islet therapy. However, a curative dose of islets within reasonably sized devices requires dense cell packing. We measured internal PO2 of implanted devices, mathematically modeled oxygen availability within devices and tested the predictions with implanted devices containing densely packed human islets. Methods: Partial pressure of oxygen (PO2) within implanted empty devices was measured by noninvasive 19F-MRS. A mathematical model was constructed, predicting internal PO2, viability and functionality of densely packed islets as a function of external PO2. Finally, viability was measured by oxygen consumption rate (OCR) in day 7 explants loaded at various islet densities. Results: In empty devices, PO2 was 12 mmHg or lower, despite successful external vascularization. Devices loaded with human islets implanted for 7 days, then explanted and assessed by OCR confirmed trends proffered by the model but viability was substantially lower than predicted. Co-localization of insulin and caspase-3 immunostaining suggested that apoptosis contributed to loss of beta cells. Discussion: Measured PO2 within empty devices declined during the first few days post-transplant then modestly increased with neovascularization around the device. Viability of islets is inversely related to islet density within devices.

Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells.

This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Translational Medicine 2017;6:1956-1962.

Derivation of Human Embryonic Stem Cells.

Embryonic stem cells (ESCs) represent a mainstay for pluripotent stem cell research and development (R&D) and provide tangible opportunities for clinical translation including cell therapies and drug discovery. Moreover, in spite of the discovery of induced pluripotent stem cells (iPSCs), ESCs are an essential reference point, against which other pluripotent cells are compared. Hence, there is an ongoing need to derive and bank quality-controlled research-grade and clinical-grade ESC lines using established and standardized methods. Here, we provide a concise, step-by-step protocol for the derivation of ESCs from human embryos. While largely based on previously reported method for clinical-grade human ESC (hESC) line derivation, the protocol is suitable for routine application, although adaptable for clinical-compliance.

Development and Validation of Noninvasive Magnetic Resonance Relaxometry for the In Vivo Assessment of Tissue-Engineered Graft Oxygenation.

Techniques to monitor the oxygen partial pressure (pO2) within implanted tissue-engineered grafts (TEGs) are critically necessary for TEG development, but current methods are invasive and inaccurate. In this study, we developed an accurate and noninvasive technique to monitor TEG pO2 utilizing proton (1H) or fluorine (19F) magnetic resonance spectroscopy (MRS) relaxometry. The value of the spin-lattice relaxation rate constant (R1) of some biocompatible compounds is sensitive to dissolved oxygen (and temperature), while insensitive to other external factors. Through this physical mechanism, MRS can measure the pO2 of implanted TEGs. We evaluated six potential MRS pO2 probes and measured their oxygen and temperature sensitivities and their intrinsic R1 values at 16.4 T. Acellular TEGs were constructed by emulsifying porcine plasma with perfluoro-15-crown-5-ether, injecting the emulsion into a macroencapsulation device, and cross-linking the plasma with a thrombin solution. A multiparametric calibration equation containing R1, pO2, and temperature was empirically generated from MRS data and validated with fiber optic (FO) probes in vitro. TEGs were then implanted in a dorsal subcutaneous pocket in a murine model and evaluated with MRS up to 29 days postimplantation. R1 measurements from the TEGs were converted to pO2 values using the established calibration equation and these in vivo pO2 measurements were simultaneously validated with FO probes. Additionally, MRS was used to detect increased pO2 within implanted TEGs that received supplemental oxygen delivery. Finally, based on a comparison of our MRS data with previously reported data, ultra-high-field (16.4 T) is shown to have an advantage for measuring hypoxia with 19F MRS. Results from this study show MRS relaxometry to be a precise, accurate, and noninvasive technique to monitor TEG pO2 in vitro and in vivo.

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids.

In this demonstration, spheroids formed from the ß-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.

Potentially immunogenic proteins expressed similarly in human embryonic stem cells and induced pluripotent stem cells.

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.

Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids.

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, ß-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.

From cell culture to a cure: pancreatic ß-cell replacement strategies for diabetes mellitus.

Numerous advances have been made in pancreatic ß-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 ß-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 ß-cells from endogenous hepatic or pancreatic tissue sources are also being investigated as a ß-cell replacement strategy. Further research is necessary to protect these transplanted or regenerated cells from diabetic autoimmune pathology.

Inhibition of DNA methyltransferases and histone deacetylases induces bone marrow-derived multipotent adult progenitor cells to differentiate into endothelial cells.

INTRODUCTION: Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular diseases and cancer. Bone marrow-derived multipotent adult progenitor cells (MAPC) have the potential to differentiate, at the single cell level, toward the three embryonic germ layers and may be the progenitors of the other tissue-specific stem cells. However, molecular mechanisms of endothelial differentiation from MAPC have not been defined. The importance of epigenetic changes such as DNA methylation and histone acetylation in gene regulatory networks during embryonic stem cell (ESC) differentiation has been documented. We postulated that endothelial cell (EC) differentiation from MAPC could be enhanced by inhibiting DNA methylation and histone deacetylation, reversing the repression of genes that specify EC fate. METHODS: MAPCs were derived from rat bone marrow and differentiated into EC by vascular endothelial growth factor (VEGF) treatment in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5'-aza-2'-deoxycytidine (aza-dC) and the histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Expression of the endothelial marker genes was assessed by real time quantitative PCR and angiogenic potential of the differentiated EC was assessed by analysis of vascular network formation on fibronectin. RESULTS: Both aza-dC and TSA induced at least a three-fold increase in the expression of the EC marker genes VE-cadherin, vWF, and Flk1. This increase was also observed in the presence of the EC differentiation inducer VEGF, suggesting that factors other than VEGF mediate the response to the epigenetic agents. Both DNMT and HDAC inhibition stimulated vascular network formation. CONCLUSION: Epigenetic therapy holds a potential in inducing self-repair, vascular tissue regeneration, controlling angiogenesis and endothelial dysfunction.

Gene expression profiles of human inner cell mass cells and embryonic stem cells.

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.

Manipulation of OCT4 levels in human embryonic stem cells results in induction of differential cell types.

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.

Development of serum-free culture systems for human embryonic stem cells.

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.

Hydrogels as artificial matrices for human embryonic stem cell self-renewal.

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.

TGFbeta inhibition of yolk-sac-like differentiation of human embryonic stem-cell-derived embryoid bodies illustrates differences between early mouse and human development.

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.

Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.

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.

Polybromo protein BAF180 functions in mammalian cardiac chamber maturation.

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.

Maintenance of pluripotency in human embryonic stem cells is STAT3 independent.

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.

Propagation and maintenance of undifferentiated human embryonic stem cells.

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 STELLAR, NANOG, and GDF3 genes are expressed in pluripotent cells and map to chromosome 12p13, a hotspot for teratocarcinoma.

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.

Spontaneous differentiation of germ cells from human embryonic stem cells in vitro.

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.