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Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

Linker histones are essential components of chromatin,but the distributions and functions of many during cellular differentiation are not well understood. Here,we show that H1.5 binds to genic and intergenic regions,forming blocks of enrichment,in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells,H1.5,but not H1.3,binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly,37% of H1.5 target genes belong to gene family clusters,groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding,H3K9me2 enrichment,and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2,increased chromatin accessibility,deregulation of gene expression,and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells. View Publication

Embryonic stem cells can replicate continuously in the absence of senescence and,therefore,are immortal in culture. Although genome stability is essential for the survival of stem cells,proteome stability may have an equally important role in stem-cell identity and function. Furthermore,with the asymmetric divisions invoked by stem cells,the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore,a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4,an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates,regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity. View Publication

Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here,we report the use of chitin-alginate 3D microfibrous scaffolds,previously developed for hPSC propagation,to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid,the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons,as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore,flow cytometry confirmed that textgreater90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines,a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons,without tumorigenesis,suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications. View Publication

BACKGROUND Strategies on the basis of doxycycline-inducible lentiviruses in mouse cells allowed the examination of mechanisms governing somatic cell reprogramming. RESULTS Using a doxycycline-inducible human reprogramming system,we identified unreported miRs enhancing reprogramming efficiency. CONCLUSION We generated a drug-inducible human reprogramming reporter system as an invaluable tool for genetic or chemical screenings. SIGNIFICANCE These cellular systems provide a tool to enable the advancement of reprogramming technologies in human cells. Reprogramming of somatic cells into induced pluripotent stem cells is achieved by the expression of defined transcription factors. In the last few years,reprogramming strategies on the basis of doxycycline-inducible lentiviruses in mouse cells became highly powerful for screening purposes when the expression of a GFP gene,driven by the reactivation of endogenous stem cell specific promoters,was used as a reprogramming reporter signal. However,similar reporter systems in human cells have not been generated. Here,we describe the derivation of drug-inducible human fibroblast-like cell lines that express different subsets of reprogramming factors containing a GFP gene under the expression of the endogenous OCT4 promoter. These cell lines can be used to screen functional substitutes for reprogramming factors or modifiers of reprogramming efficiency. As a proof of principle of this system,we performed a screening of a library of pluripotent-enriched microRNAs and identified hsa-miR-519a as a novel inducer of reprogramming efficiency. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

Emerging studies suggested that murine podoplanin-positive monocytes (PPMs) are involved in lymphangiogenesis. The goal of this study was to demonstrate the therapeutic lymphangiogenesis of human PPMs by the interaction with platelets. Aggregation culture of human peripheral blood mononuclear cells (PBMCs) resulted in cellular aggregates termed hematospheres. During 5-day culture,PPMs expanded exponentially and expressed several lymphatic endothelial cell-specific markers including vascular endothelial growth factor receptor (VEGFR)-3 and well-established lymphangiogenic transcription factors. Next,we investigated the potential interaction of PPMs with platelets that had C-type lectin-like receptor-2 (CLEC-2),a receptor of podoplanin. In vitro coculture of PPMs and platelets stimulated PPMs to strongly express lymphatic endothelial markers and upregulate lymphangiogenic cytokines. Recombinant human CLEC-2 also stimulated PPMs through Akt and Erk signaling. Likewise,platelets in coculture with PPMs augmented secretion of a lymphangiogenic cytokine,interleukin (IL)-1-β,via podoplanin/CLEC-2 axis. The supernatant obtained from coculture was able to enhance the migration,viability,and proliferation of lymphatic endothelial cell. Local injection of hematospheres with platelets significantly increased lymphatic neovascularization and facilitated wound healing in the full-thickness skin wounds of nude mice. Cotreatment with PPMs and platelets augments lymphangiogenesis through podoplanin/CLEC-2 axis,which thus would be a promising novel strategy of cell therapy to treat human lymphatic vessel disease. View Publication

In metazoans,the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes,by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific,while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps,we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover,cLADs are depleted of synteny breakpoints,pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically,the overall sequence conservation is low for cLADs. Instead,cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this A/T rule" in embryonic stem cells� View Publication

Retinitis pigmentosa,other inherited retinal diseases,and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here,we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx+ photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells,facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. STEM CELLS2013;31:408–414 View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication