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We measured the dynamics of an essential epigenetic modifier,HP1β,in human cells at different stages of differentiation using Fluorescence Recovery After Photobleaching (FRAP). We found that HP1β mobility is similar in human embryonic stem cells (hES) and iPS cells where it is more mobile compared to fibroblasts; HP1β is less mobile in senescent fibroblasts than in young (dividing) fibroblasts. Introduction of reprogramming factors"� View Publication

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells,called induced pluripotent stem cells (iPSCs),can be an unlimited source of specialized cell types for the body. Thus,autologous somatic cell replacement therapy becomes possible,as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited,and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. View Publication

Human stem cells are promising sources for bladder regeneration. Among several possible sources,pluripotent stem cells are the most fascinating because they can differentiate into any cell type,and proliferate limitlessly in vitro. Here,we developed a protocol for differentiation of human pluripotent stem cells (hPSCs) into bladder urothelial cells (BUCs) under a chemically defined culture system. We first differentiated hPSCs into definitive endoderm (DE),and further specified DE cells into BUCs by treating retinoic acid under a keratinocyte-specific serum free medium. hPSC-derived DE cells showed significantly expressed DE-specific genes,but did not express mesodermal or ectodermal genes. After DE cells were specified into BUCs,they notably expressed urothelium-specific genes such as UPIb,UPII,UPIIIa,P63 and CK7. Immunocytochemistry showed that BUCs expressed UPII,CK8/18 and P63 as well as tight junction molecules,E-CADHERIN and ZO-1. Additionally,hPSCs-derived BUCs exhibited low permeability in a FITC-dextran permeability assay,indicating BUCs possessed the functional units of barrier on their surfaces. However,BUCs did not express the marker genes of other endodermal lineage cells (intestine and liver) as well as mesodermal or ectodermal lineage cells. In summary,we sequentially differentiated hPSCs into DE and BUCs in a serum- and feeder-free condition. Our differentiation protocol will be useful for producing cells for bladder regeneration and studying normal and pathological development of the human bladder urothelium in vitro. View Publication

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease,as well as for future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture,differentiation,isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 d),isolation (1 d) and 4 or 10 d of expansion of ECs and pericytes,respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon coculture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs. View Publication

BACKGROUND Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However,the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. METHODS hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st,3rd,5th,8th,10th,and 15th passage,the differentiation of MSCs into osteogenic,chondrogenic,and adipogenic cells was detected,and MTT,surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0,8,and 15. The telomerase activity of hUC-MSCs at passage 1-15 was analyzed. RESULTS Flow cytometry analysis showed that very high expression was detected for CD105,CD73,and CD90 and very low expression for CD45,CD34,CD14,CD79a,and HLA-DR. MSCs could differentiate into osteocytes,chondrocytes,and adipocytes in vitro. There was no obvious chromosome elimination,displacement,or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further,no tumors formed in rats injected with hUC-MSCs (P15) cultured in serum-free and in serum-containing conditions. CONCLUSION Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium,these cells are suitable for cell therapy. View Publication

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation,microcephaly,and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4),Artemis,and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders,we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity,but not Artemis deficiency,were associated with markedly reduced reprogramming efficiency,which could be partially rescued by genetic complementation. Moreover,we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest,particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-,but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming,with a major role for LIG4 and DNA-PKcs and a minor,if any,for Artemis. View Publication

Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs,we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post-transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR-203 has been extensively studied in human tumors,but has not been characterized in hESCs. We show that miR-203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles,controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore,aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes. While salt-induced colloidal nanosphere aggregates have produced robust SERS signals,their variability in aggregate size contributes significantly to poor SERS signal reproducibility,which can complicate their use in in vitro cellular studies. Such systems often also lack reproducibility in spectral measurements between different nanoparticle clusters. Preaggregation of colloids via linkers followed by surface functionalization with reporter molecules results in the linker occupying valuable SERS hotspot volume which could otherwise be utilized by additional reporter molecules. Ideally,both functionalities should be obtained from a single molecule. Here,we report the use of 3,5-dimercaptobenzoic acid,a single multifunctional molecule that creates SERS hotspots via the controlled aggregation of nanoparticles,and also reports pH values. We show that 3,5-dimercaptobenzoic acid bound to Au nanospheres results in an excellent pH nanoprobe,producing very robust,and highly reproducible SERS signals that can report pH across the entire physiological range with excellent pH resolution. To demonstrate the efficacy of our novel pH reporters,these probes were also used to image both the particle and pH distribution in the cytoplasm of human induced pluripotent stem cells (hiPSCs). View Publication

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however,differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4,colonies reproducibly differentiated to an outer trophectoderm-like ring,an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development. View Publication

Generation of surrogate sources of insulin-producing β-cells remains a goal of diabetes therapy. While most efforts have been directed at differentiating embryonic or induced pluripotent stem (iPS) cells into β-like-cells through endodermal progenitors,we have shown that gut endocrine progenitor cells of mice can be differentiated into glucose-responsive,insulin-producing cells by ablation of transcription factor Foxo1. Here we show that FOXO1 is present in human gut endocrine progenitor and serotonin-producing cells. Using gut organoids derived from human iPS cells,we show that FOXO1 inhibition using a dominant-negative mutant or lentivirus-encoded small hairpin RNA promotes generation of insulin-positive cells that express all markers of mature pancreatic β-cells,release C-peptide in response to secretagogues and survive in vivo following transplantation into mice. The findings raise the possibility of using gut-targeted FOXO1 inhibition or gut organoids as a source of insulin-producing cells to treat human diabetes. View Publication