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Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study,we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4,hSox2,hKlf4,and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci,a hallmark of DM1,were detected in DM1 iPSCs,neural stem cells (NSCs),and terminally differentiated neurons and astrocytes. In conclusion,we have successfully established disease-specific human DM1 iPSC lines,NSCs,and neuronal lineages with pathognomonic intranuclear RNA foci,which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development. View Publication

We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs),partially reprogrammed cells,somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over approximately 10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free,enriched to 95%-99% purity with textgreater80% survival,and had normal transcriptional profiles,differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells. View Publication

Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However,the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles,we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct,optimized for high co-expression of OCT4,SOX2,KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades,such as transforming growth factor $\$(SB431542),MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin),in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design,signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol,we could derive iPSCs from patient fibroblasts,which were impermissive to classical reprogramming efforts,and from a patient suffering from familial platelet disorder. Thus,our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs,reflects a big step towards therapeutic and broad scientific application of iPSCs,even in previously unfeasible settings. View Publication

Human pluripotent stem cells are a powerful tool for modeling brain development and disease. The human cortex is composed of two major neuronal populations: projection neurons and local interneurons. Cortical interneurons comprise a diverse class of cell types expressing the neurotransmitter GABA. Dysfunction of cortical interneurons has been implicated in neuropsychiatric diseases,including schizophrenia,autism,and epilepsy. Here,we demonstrate the highly efficient derivation of human cortical interneurons in an NKX2.1::GFP human embryonic stem cell reporter line. Manipulating the timing of SHH activation yields three distinct GFP+ populations with specific transcriptional profiles,neurotransmitter phenotypes,and migratory behaviors. Further differentiation in a murine cortical environment yields parvalbumin- and somatostatin-expressing neurons that exhibit synaptic inputs and electrophysiological properties of cortical interneurons. Our study defines the signals sufficient for modeling human ventral forebrain development in vitro and lays the foundation for studying cortical interneuron involvement in human disease pathology. View Publication

Label-retaining cancer cells (LRCCs) represent a novel population of stem-like cancer cells exhibiting slow cycling,chemoresistance and tumor-initiating capacities; however,their properties remain unclear,and approaches to eradicate LRCCs remain elusive. Here,we report that colon cancer cells with high fluorescent intensity,referred to as LRCCs,have the greatest cancer stem cell (CSC)-like capacities and that they preferentially express CSC markers and stemness-related genes. Moreover,we found that Lgr5,which has been reported to be a marker of rapid cycling CSCs,is almost negatively expressed in LRCCs but that its expression is gradually increased in the differentiation process of LRCCs. Interestingly,we found that LRCCs are especially sensitive to the pro-apoptotic effect of IFN-γ treatment both in vitro and in vivo because LRCCs possess higher IFN-γR levels compared with non-LRCCs,which results in the upregulation of the apoptosis pathway after IFN-γ treatment. Furthermore,we found that IFN-γ shows synergistic effects with the conventional anticancer drug Oxaliplatin to eliminate both LRCCs and non-LRCCs. In conclusion,this is the first study to suggest that LRCCs,as a distinct tumor-initiating population,can be selectively eradicated by IFN-γ,which may provide a novel therapeutic strategy for colon cancer treatment. View Publication

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However,molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further,Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway,comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3,were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover,inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last,radiation treatment of PN GSCs up-regulated Mes-associated markers and down-regulated PN-associated markers,whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together,our data suggest that two subtypes of GSCs,harboring distinct metabolic signaling pathways,represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3-mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature. View Publication

A satisfactory in vitro model of vocal fold mucosa does not exist,thus precluding a systematic,controlled study of vocal fold biology and biomechanics. We sought to create a valid,reproducible three-dimensional (3D) in vitro model of human origin of vocal fold mucosa of human origin. We hypothesized that coculture of human embryonic stem cell (hESC)-derived simple epithelial cells with primary vocal fold fibroblasts under appropriate conditions would elicit morphogenesis of progenitor cells into vocal fold epithelial-like cells and creation of a basement membrane. Using an in vitro prospective study design,hESCs were differentiated into cells that coexpressed the simple epithelial cell marker,keratin 18 (K18),and the transcription factor,p63. These simple epithelial cells were cocultured with primary vocal fold fibroblasts seeded in a collagen gel scaffold. The cells were cultured for 3 weeks in a keratinocyte medium at an air–liquid interface. After that time,the engineered mucosa demonstrated a stratified,squamous epithelium and a continuous basement membrane recapitulating the key morphologic and phenotypic characteristics of native vocal fold mucosa. hESC-derived epithelial cells exhibited positive staining for vocal fold stratified,squamous epithelial markers,keratin 13 (K13) and 14 (K14),as well as tight junctions,adherens junctions,gap junctions,and desmosomes. Despite the presence of components critical for epithelial structural integrity,the epithelium demonstrated greater permeability than native tissue indicating compromised functional integrity. While further work is warranted to improve functional barrier integrity,this study demonstrates that hESC-derived epithelial progenitor cells can be engineered to create a replicable 3D in vitro model of vocal fold mucosa featuring a multilayered,terminally differentiated epithelium. View Publication

Definitive endoderm can be derived from human embryonic stem cells using low serum medium with cytokines involved in the epithelial-to-mesenchymal transition,including Activin A and Wnt3A. The purpose of this study was to develop an improved protocol that permits the induction of definitive endoderm while avoiding the high rate of cell death that often occurs with existing protocols. By including insulin and other nutrients,we demonstrate that cell viability can be preserved throughout differentiation. In addition,modifying a matrigel sandwich method previously reported to induce precardiac mesoderm allows for enhanced endodermal differentiation based on expression of endoderm-associated genes. The morphological and migratory characteristics of cells cultured by the technique,as well as gene expression patterns,indicate that the protocol can emulate key events in gastrulation towards the induction of definitive endoderm. View Publication

PURPOSE FK506-binding protein like (FKBPL) and its peptide derivative,AD-01,have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here,we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC). EXPERIMENTAL DESIGN Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75),primary patient samples,and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays,quantitative PCR (qPCR),and immunofluorescence. RESULTS AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers,Nanog,Oct4,and Sox2,were also significantly reduced. Furthermore,we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor,DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally,FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs,highlighting a role for endogenous FKBPL in stem cell signaling. CONCLUSIONS AD-01 has dual antiangiogenic and anti-BCSC activity,which will be advantageous as this agent enters clinical trial. View Publication

Lymphocyte activation is regulated by costimulatory and inhibitory receptors,of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably,it remains unclear how HVEM functions with each of its ligands during immune responses. In this study,we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT,Lymphotoxin-α,or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2,resulting in increased IFN-γ and TNF-α secretion,and tumor cell-expressed HVEM activated CD160 in a human NK cell line,causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast,HVEM activation of BTLA reduced cytolysis of target cells. Together,our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA. View Publication

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy,drug screening and toxicity testing of neural degenerative diseases. However,the molecular regulation of their proliferation and apoptosis,which needs to be revealed before clinical application,is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here,we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells,hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently,global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically,a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression,revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover,forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly,we found that paraquat,a neurotoxin,not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs,indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably,inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively,miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis. View Publication

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling,which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs,suggesting that they may influence self-renewal. To test this hypothesis,we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation,whereas SPRY4 KD enhanced survival. In both cases,after KD the cells were able to differentiate into cells of the three germ layers,although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage,explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs. View Publication