In Vivo CaV3 Channel Inhibition Promotes Maturation of Glucose-Dependent Ca2+ Signaling in Human iPSC-Islets.

CaV3 channels are ontogenetically downregulated with the maturation of certain electrically excitable cells, including pancreatic ß cells. Abnormally exaggerated CaV3 channels drive the dedifferentiation of mature ß cells. This led us to question whether excessive CaV3 channels, retained mistakenly in engineered human-induced pluripotent stem cell-derived islet (hiPSC-islet) cells, act as an obstacle to hiPSC-islet maturation. We addressed this question by using the anterior chamber of the eye (ACE) of immunodeficient mice as a site for recapitulation of in vivo hiPSC-islet maturation in combination with intravitreal drug infusion, intravital microimaging, measurements of cytoplasmic-free Ca2+ concentration ([Ca2+]i) and patch clamp analysis. We observed that the ACE is well suited for recapitulation, observation and intervention of hiPSC-islet maturation. Intriguingly, intraocular hiPSC-islet grafts, retrieved intact following intravitreal infusion of the CaV3 channel blocker NNC55-0396, exhibited decreased basal [Ca2+]i levels and increased glucose-stimulated [Ca2+]i responses. Insulin-expressing cells of these islet grafts indeed expressed the NNC55-0396 target CaV3 channels. Intraocular hiPSC-islets underwent satisfactory engraftment, vascularization and light scattering without being influenced by the intravitreally infused NNC55-0396. These data demonstrate that inhibiting CaV3 channels facilitates the maturation of glucose-activated Ca2+ signaling in hiPSC-islets, supporting the notion that excessive CaV3 channels as a developmental error impede the maturation of engineered hiPSC-islet insulin-expressing cells.

Intracameral Microimaging of Maturation of Human iPSC Derivatives into Islet Endocrine Cells.

We exploited the anterior chamber of the eye (ACE) of immunodeficient mice as an ectopic site for both transplantation and microimaging of engineered surrogate islets from human induced pluripotent stem cells (hiPSC-SIs). These islets contained a majority of insulin-expressing cells, positive or negative for PDX1 and NKX6.1, and a minority of glucagon- or somatostatin-positive cells. Single, non-aggregated hiPSC-SIs were satisfactorily engrafted onto the iris. They underwent gradual vascularization and progressively increased their light scattering signals, reflecting the abundance of zinc-insulin crystal packaged inside mature insulin secretory granules. Intracameral hiPSC-SIs retrieved from recipients showed enhanced insulin immunofluorescence in correlation with the parallel increase in overall vascularization and light backscattering during the post-transplantation period. This approach enables longitudinal, nondestructive and intravital microimaging of cell fates, engraftment, vascularization and mature insulin secretory granules of single hiPSC-SI grafts, and may offer a feasible and reliable means to screen compounds for promoting in vivo hiPSC-SI maturation.

Techniques in embryoid body formation from human pluripotent stem cells.

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels, under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm, mesoderm, and endoderm. In this chapter, we describe techniques for generation of EBs from human pluripotent stem cells. Once formed, the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research, drug discovery/development, and cellular transplantation therapy.

Endogenous fluorescence signatures in living pluripotent stem cells change with loss of potency.

The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications.

Induced pluripotent reprogramming from promiscuous human stemness related factors.

Ectopic expression of pluripotency gene sets provokes nuclear reprogramming in permissive somatic tissue environments generating induced pluripotent stem (iPS) cells. The evolutionary conserved function of stemness orthologs was here tested through interspecies transduction. A spectrum of HIV-based lentiviral vectors was designed, and point mutations in the HIV-1 capsid region identified for efficient infectivity and expanded trans-species tropism. Human pluripotent gene sequences, OCT3/4, SOX2, KLF4 and c-MYC, packaged into engineered lentiviral expression vectors achieved consistent expression in non-human fibroblasts. Despite variation in primary amino-acid sequence between species, introduction of human pluripotent genes produced cell lines with embryonic stem cell-like morphology. Transduced fibroblasts differentiated in vitro into all three germ layers according to gastrulation gene expression profiles, and formed in vivo teratoma with multi-lineage potential. Reprogrammed progeny incorporated into non-human morula to produce blastomeres capable of developing into chimeric embryos with competent organogenesis. This model system establishes a prototypic approach to examine consequences of human stemness factors induced reprogramming in the context of normal embryonic development, exploiting non-human early stage embryos. Thus, ectopic xeno-transduction across species unmasks the promiscuous nature of stemness induction, suggesting evolutionary selection of core processes for somatic tissue reprogramming.

Alpha interferon enhances TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells.

Dominant, constitutively expressed antiretroviral factors, including TRIM5alpha and APOBEC3 proteins, are distinguished from the conventional innate immune systems and are classified as intrinsic immunity factors. Here, we demonstrate that interferon alpha (IFN-alpha) treatment upregulates TRIM5alpha mRNA in rhesus monkey cells, which correlates with the enhanced TRIM5alpha-mediated pre- and postintegration blocks of human immunodeficiency virus replication. In human cells, IFN-alpha increases the levels of TRIM5alpha mRNA, resulting in enhanced antiviral activity against N-tropic murine leukemia virus infection. These observations indicate that the TRIM5alpha-mediated antiviral effects can be orchestrated by the conventional innate immune response. It is conceivable that TRIM5alpha plays an essential role in controlling both the initial retroviral exposure and the subsequent viral dissemination in vivo.

The RAS/Raf1/MEK/ERK signaling pathway facilitates VSV-mediated oncolysis: implication for the defective interferon response in cancer cells.

Vesicular stomatitis virus (VSV) can replicate in malignant cells more efficiently than in normal cells. Although the selective replication appears to be caused by defects in the interferon (IFN) system in malignant cells, the mechanisms which render these cells less responsive to IFN remain poorly understood. Here we present evidence that an activated RAS/Raf1/MEK/ERK pathway plays a critical role in the defects. NIH 3T3 or human primary cells stably expressing active RAS or Raf1 were rapidly killed by VSV. Although IFNalpha treatment no longer protected the RAS- or Raf1-overexpressing cells from VSV infection, responsiveness to IFNalpha was restored following treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Similarly, human cancer-derived cell lines became more responsive to IFNalpha in conjunction with U0126 treatment. Intriguingly, dual treatment with both IFNalpha and U0126 severely reduced the levels of viral RNAs in the infected cells. Moreover, cancer cells showed defects in inducing an IFNalpha-responsive factor, MxA, which is known to block VSV RNA synthesis, and U0126 restored the MxA expression. Our observations suggest that activation of the extracellular signal-regulated protein kinase (ERK) signaling leads to the defect in IFNalpha-mediated upregulation of MxA protein, which facilitates VSV oncolysis. In view of the fact that 30% of all cancers have constitutive activation of the RAS/Raf1/MEK/ERK pathway, VSV would be an ideal oncolytic virus for targeting such cancers.