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Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types.

Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of NKX2-1 is required to specify definitive endoderm to respiratory endoderm. However, the transcriptional regulation of NKX2-1 is not fully understood. Here we demonstrate that aside from specifying undifferentiated human embryonic stem cell (hESC) to definitive endoderm, high concentrations of Activin-A are also necessary and sufficient to induce hESC-derived definitive endodermal progeny to a FOXA2/NKX2-1/GATA6/PAX9 positive respiratory epithelial fate. Activin-A directly mediates the induction of NKX2-1 by interacting with ALK4, leading to phosphorylation of SMAD2, which binds directly to the NKX2-1 promoter and activates its expression. Activin-A can be replaced by GDF11 but not transforming growth factor β1. Addition of Wnt3a, SHH, FGF2, or BMP4 failed to induce NKX2-1. These results suggest that direct binding of Activin-A-responsive SMAD2 to the NKX2-1 promoter plays essential role during respiratory endoderm specification.

The potential of using bone marrow (BM)-derived human stromal cells for ex vivo gene therapy of hemophilia A was evaluated. BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (Mo-MuLV) retroviral vector that contained the B domain-deleted human factor VIII (FVIIIdeltaB) cDNA. This FVIII-retroviral vector was pseudotyped with the gibbon ape leukemia virus envelope (GALV-env) to attain higher transduction efficiencies. Using optimized transduction methods, high in vitro FVIII expression levels of 700 to 2500 mU of FVIII/10(6) cells per 24 hr were achieved without selective enrichment of the transduced BM stromal cells. After xenografting of 1.5-3 x 106 engineered BM stromal cells into the spleen of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, human plasma FVIII levels rose to 13 +/- 4 ng/ml but declined to basal levels by 3 weeks postinjection because of promoter inactivation. About 10% of these stromal cells engrafted in the spleen and persisted for at least 4 months after transplantation in the absence of myeloablative conditioning. No human BM stromal cells could be detected in other organs. These findings indicate that retroviral vector-mediated gene therapy using engineered BM stromal cells may lead to therapeutic levels of FVIII in vivo and that long-term engraftment of human BM stromal cells was achieved in the absence of myeloablative conditioning and without neo-organs. Hence, BM stromal cells may be useful for gene therapy of hemophilia A, provided prolonged expression can be achieved by using alternative promoters.

Hemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and predisposes to spontaneous bleeding that can be life-threatening or lead to chronic disabilities. It is well suited for gene therapy because a moderate increase in plasma FVIII concentration has therapeutic effects. Improved retroviral vectors expressing high levels of human FVIII were pseudotyped with the vesicular stomatitis virus G glycoprotein, were concentrated to high-titers (10(9)-10(10) colony-forming units/ml), and were injected intravenously into newborn, FVIII-deficient mice. High-levels (>/=200 milliunits/ml) of functional human FVIII production could be detected in 6 of the 13 animals, 4 of which expressed physiologic or higher levels (500-12,500 milliunits/ml). Five of the six expressers produced FVIII and survived an otherwise lethal tail-clipping, demonstrating phenotypic correction of the bleeding disorder. FVIII expression was sustained for >14 months. Gene transfer occurred into liver, spleen, and lungs with predominant FVIII mRNA expression in the liver. Six of the seven animals with transient or no detectable human FVIII developed FVIII inhibitors (7-350 Bethesda units/ml). These findings indicate that a genetic disease can be corrected by in vivo gene therapy using retroviral vectors.

Attempts to develop an ex vivo gene therapy strategy for hemophilia A, using either primary T cells or bone marrow (BM) stem/progenitor cells have been unsuccessful, due to the inability of these cell types to express coagulation factor VIII (FVIII). As an alternative, we evaluated the potential of BM-derived stromal cells which can be readily obtained and expanded in vitro. Human and murine BM stromal cells were transduced with an intron-based Moloney murine leukemia virus (MoMLV) retroviral vector expressing a B-domain-deleted human factor VIII cDNA (designated as MFG-FVIIIdeltaB). Transduction efficiencies were increased 10- to 15-fold by phosphate depletion and centrifugation, which obviated the need for selective enrichment of the transduced BM stromal cells. This resulted in high FVIII expression levels in transduced human (180 +/- 4 ng FVIII/10[6] cells per 24 hr) and mouse (900 +/- 130 ng FVIII/10[6] cells per 24 hr) BM stromal cells. Pseudotyping of the MFG-FVIIIdeltaB retroviral vectors with the gibbon ape leukemia virus envelope (GALV-env) resulted in significantly higher transduction efficiencies (100 +/- 20%) and FVIII expression levels (390 +/- 10 ng FVIII/10[6] cells per 24 hr) in transduced human BM stromal cells than with standard amphotropic vectors. This difference in transduction efficiency correlated with the higher titer of the GALV-env pseudotyped viral vectors and with the higher GALV receptor (GLVR-1) versus amphotropic receptor (GLVR-2) mRNA expression levels in human BM stromal cells. These findings demonstrate the potential of BM stromal cells for gene therapy in general and hemophilia A in particular.