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The mechanisms underlying human germ cell development are largely unknown,partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here,we studied NANOS3 and DAZL,which have critical roles in germ cell development in several species,via their over expression in human embryonic stem cells using global transcriptional analysis,in vitro germ cell differentiation,and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition,we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL,our results suggest a post-transcriptional regulation mechanism in hES cells. In addition,we found that DAZL suppressed the translation of OCT4,and affected the transcription of several genes associated with germ cells,cell cycle arrest,and cell migration. Furthermore,DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. View Publication

A new method of spatially controlled gene regulation in 3D-cultured human embryonic stem cells is developed using hollow gold nanoshells (HGNs) and near-infrared (NIR) light. Targeted cell(s) are discriminated from neighboring cell(s) by focusing NIR light emitted from a two-photon microscope. Irradiation of cells that have internalized HGNs releases surface attached siRNAs and leads to concomitant gene downregulation. View Publication

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs,we identified a mitochondrial protein,CHCHD2,whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria,resulting in suppression of the activity of the TGFβ signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies. View Publication

Understanding the molecular mechanisms that underlie neurodegenerative disorders has been hampered by a lack of readily available model systems that replicate the complexity of the human disease. Recent advances in stem cell technology have facilitated the derivation of patient-specific stem cells from a variety of differentiated cell types. These induced pluripotent stem cells (iPSCs) are attractive disease models since they can be grown and differentiated to produce large numbers of disease-relevant cell types. However,most iPSC lines are derived in advance of,and without the benefit of,neuropathological confirmation of the donor - the gold standard for many disease classifications and measurement of disease severity. While others have reported the generation of autopsy-confirmed iPSC lines from patient explants,these methods require outgrowth of cadaver tissue,which require additional time and is often only successul 50% of the time. Here we report the rapid generation of autopsy-confirmed iPSC lines from peripheral blood mononuclear cells (PBMCs) drawn postmortem. Since this approach doesn't require the propagation of previously frozen cadaver tissue,iPSC can be rapidly and efficiently produced from patients with autopsy-confirmed pathology. These matched iPSC-derived patient-specific neurons and postmortem brain tissue will support studies of specific mechanisms that drive the pathogenesis of neurodegenerative diseases. View Publication

The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However,generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)-derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons,which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology,we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure,function,and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. View Publication

Human embryonic stem cells can replicate indefinitely while maintaining their undifferentiated state and,therefore,are immortal in culture. This capacity may demand avoidance of any imbalance in protein homeostasis (proteostasis) that would otherwise compromise stem cell identity. Here we show that human pluripotent stem cells exhibit enhanced assembly of the TRiC/CCT complex,a chaperonin that facilitates the folding of 10% of the proteome. We find that ectopic expression of a single subunit (CCT8) is sufficient to increase TRiC/CCT assembly. Moreover,increased TRiC/CCT complex is required to avoid aggregation of mutant Huntingtin protein. We further show that increased expression of CCT8 in somatic tissues extends Caenorhabditis elegans lifespan in a TRiC/CCT-dependent manner. Ectopic expression of CCT8 also ameliorates the age-associated demise of proteostasis and corrects proteostatic deficiencies in worm models of Huntington's disease. Our results suggest proteostasis is a common principle that links organismal longevity with hESC immortality. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

Human embryonic stem cell (hESC) line chHES-480 was derived from abnormal blastocyst diagnosed with adrenoleukodystrophy (ALD) after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-480 cell line carried a hemizygous missense mutation c.1825GtextgreaterA(p.Glu609Lys) of ABCD1 gene. Characteristic tests proved that the chHES-480 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. View Publication

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies,many of which will be aimed at aged patients. To explore the impact of age on iPSC quality,we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age,which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells,which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age,and all iPSC lines analyzed carry at least one gene-disrupting mutation,several of which have been associated with cancer or dysfunction. Unexpectedly,elderly donors (textgreater90 yrs) harbor fewer mutations than predicted,likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology. View Publication

Success in the differentiating human embryonic stem cells (hESCs) into insulin-secreting β cells raises new hopes for diabetes treatment. In this work,we demonstrated the feasibility of developing islet organoids from hESCs within biomimetic 3D scaffolds. We showed that such a 3D microenvironment is critical to the generation of pancreatic endoderm and endocrine from hESCs. The organoids formed consisted of pancreatic α,β,δ,and pancreatic polypeptide (PP) cells. A high-level co-expression of PDX1,NKX6.1,and NGN3 in these cells suggests the characteristics of pancreatic β cells. More importantly,most insulin-secreting cells generated did not express glucagon,somatostatin,or PP. The expression of mature β cell marker genes such as Pdx1,Ngn3,Insulin,MafA,and Glut2 was detected in these 3D-induced cell clusters. A high-level expression of C-peptide confirmed the de novo endogenous insulin production in these 3D induced cells. Insulin-secretory granules,an indication of β cell maturity,were detected in these cells as well. Glucose challenging experiments suggested that these cells are sensitive to glucose levels due to their elevated maturity. Exposing the cells to a high concentration of glucose induced a sharp increase in insulin secretion. View Publication

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

Importance Human motor neurons may be reliably derived from induced pluripotent stem cells (iPSCs). In vivo transplant studies of human iPSCs and their cellular derivatives are essential to gauging their clinical utility. Objective To determine whether human iPSC-derived motor neurons can engraft in an immunodeficient mouse model of sciatic nerve injury. Design,Setting,and Subjects This nonblinded interventional study with negative controls was performed at a biomedical research institute using an immunodeficient,transgenic mouse model. Induced pluripotent stem cell-derived motor neurons were cultured and differentiated. Cells were transplanted into 32 immunodeficient mice with sciatic nerve injury aged 6 to 15 weeks. Tissue analysis was performed at predetermined points after the mice were killed humanely. Animal experiments were performed from February 24,2015,to May 2,2016,and data were analyzed from April 7,2015,to May 27,2016. Interventions Human iPSCs were used to derive motor neurons in vitro before transplant. Main Outcomes and Measures Evidence of engraftment based on immunohistochemical analysis (primary outcome measure); evidence of neurite outgrowth and neuromuscular junction formation (secondary outcome measure); therapeutic effect based on wet muscle mass preservation and/or electrophysiological evidence of nerve and muscle function (exploratory end point). Results In 13 of the 32 mice undergoing the experiment,human iPSC-derived motor neurons successfully engrafted and extended neurites to target denervated muscle. Human iPSC-derived motor neurons reduced denervation-induced muscular atrophy (mean [SD] muscle mass preservation,54.2% [4.0%]) compared with negative controls (mean [SD] muscle mass preservation,33.4% [2.3%]) (P = .04). No electrophysiological evidence of muscle recovery was found. Conclusions and Relevance Human iPSC-derived motor neurons may have future use in the treatment of peripheral motor nerve injury,including facial paralysis. Level of Evidence NA. View Publication