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Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results,NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2,LMX1A,TH,GIRK2,PITX3,EN1,NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue,with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation,the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. View Publication

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm,which is manifested by rosette formation,with consecutive differentiation into neural progenitors and early glial-like cells. In this study,we examined the involvement of early neural markers - OTX2,PAX6,Sox1,Nestin,NR2F1,NR2F2,and IRX2 - in the onset of rosette formation,during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation,which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation,when rosettes comprise no more than 3-5 cells,and that its expression precedes that of established markers of early neuronal differentiation. Importantly,the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly,we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro,and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. ?? 2013 Elsevier B.V. View Publication

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS),as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear,with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed,and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased,leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α,suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6,and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. View Publication

In the past decade,tissue microarray (TMA) technology has evolved as an innovative tool for high-throughput proteomics analysis and mainly for biomarker validation. Similarly,enormous amount of data can be obtained from the cell line macroarray (CLMA) technology,which developed from the TMA using formalin-fixed,paraffin-embedded cell pellets. Here,we applied CLMA technology in stem cell research and in particular to identify bona fide neogenerated human induced pluripotent stem cell (hiPSC) clones suitable for down the line differentiation. All hiPSC protocols generate tens of clones,which need to be tested to determine genetically stable cell lines suitable for differentiation. Screening methods generally rely on fluorescence-activated cell sorting isolation and coverslip cell growth followed by immunofluorescence; these techniques could be cumbersome. Here,we show the application of CLMA to identify neogenerated pluripotent cell colonies and neuronal differentiated cell products. We also propose the use of the automated image analyzer,TissueQuest,as a reliable tool to quickly select the best clones,based upon the level of expression of multiple pluripotent biomarkers. 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

BACKGROUND Human microRNAs (miRNAs) have diverse functions in biology,and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS To examine whether there were any changes in mutation status in cells treated with 520d-5p,we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS 520d-5p induced a shift towards a wild type or non-malignant phenotype,which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further,520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS Cancer cell genomic mutations cannot be repaired in most contexts. However,these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine. View Publication

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However,methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately,these methods entail numerous challenges,including poor efficiency,variable cell type heterogeneity,and lengthy,expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol,these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid,efficient,and scalable manner (Wang et al.,2017). In this manuscript,we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs,and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) obtained from a 62-year-old female with familial hypertrophic cardiomyopathy (HCM). PBMCs were reprogrammed to a pluripotent state following transfection with non-integrative episomal vectors carrying reprogramming factors OCT4,SOX2,LIN28,KLF4 and L-MYC. iPSCs were shown to express pluripotency markers,possess trilineage differentiation potential,carry rare variants identified in DNA isolated directly from the patient's whole blood,have a normal karyotype and no longer carry episomal vectors for reprogramming. This line is a useful resource for identifying unknown genetic causes of HCM. View Publication