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.

A Report from a Workshop of the International Stem Cell Banking Initiative, Held in Collaboration of Global Alliance for iPSC Therapies and the Harvard Stem Cell Institute, Boston, 2017.

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.

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.

Establishment of leukemia inhibitory factor (LIF)-independent iPS cells with potentiated Oct4.

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 90 days 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.

Derivation of LIF-independent mouse iPS cells with modified Oct4.

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.

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.

Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

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.

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.

Establishment of LIF-dependent human iPS cells closely related to basic FGF-dependent authentic iPS cells.

Human induced pluripotent stem cells (iPSCs) can be divided into a leukemia inhibitory factor (LIF)-dependent naïve type and a basic fibroblast growth factor (bFGF)-dependent primed type. Although the former are more undifferentiated than the latter, they require signal transduction inhibitors and sustained expression of the transgenes used for iPSC production. We used a transcriptionally enhanced version of OCT4 to establish LIF-dependent human iPSCs without the use of inhibitors and sustained transgene expression. These cells belong to the primed type of pluripotent stem cell, similar to bFGF-dependent iPSCs. Thus, the particular cytokine required for iPSC production does not necessarily define stem cell phenotypes as previously thought. It is likely that the bFGF and LIF signaling pathways converge on unidentified OCT4 target genes. These findings suggest that our LIF-dependent human iPSCs could provide a novel model to investigate the role of cytokine signaling in cellular reprogramming.

Efficient iPS cell production with the MyoD transactivation domain in serum-free culture.

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.

Radical acceleration of nuclear reprogramming by chromatin remodeling with the transactivation domain of MyoD.

Induced pluripotent stem cells (iPSCs) can be created by reprogramming differentiated cells through introduction of defined genes, most commonly Oct4, Sox2, Klf4, and c-Myc (OSKM). However, this process is slow and extremely inefficient. Here, we demonstrate radical acceleration of iPSC creation with a fusion gene between Oct4 and the powerful transactivation domain (TAD) of MyoD (M(3)O). Transduction of M(3) O as well as Sox2, Klf4, and c-Myc into fibroblasts effectively remodeled patterns of DNA methylation, chromatin accessibility, histone modifications, and protein binding at pluripotency genes, raising the efficiency of making mouse and human iPSCs more than 50-fold in comparison to OSKM. These results identified that one of the most critical barriers to iPSC creation is poor chromatin accessibility and protein recruitment to pluripotency genes. The MyoD TAD has a capability of overcoming this problem. Our approach of fusing TADs to unrelated transcription factors has far-reaching implications as a powerful tool for transcriptional reprogramming beyond application to iPSC technology.

Human embryonic and rat adult stem cells with primitive endoderm-like phenotype can be fated to definitive endoderm, and finally hepatocyte-like cells.

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.

Liver xeno-repopulation with human hepatocytes in Fah-/-Rag2-/- mice after pharmacological immunosuppression.

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.

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.