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The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response,but an in-depth exploration of response levels is lacking. Herein,using an established method of airway epithelial generation from hPSCs,we show that hPSC-derived epithelial cells are able to up-regulate expression of TNF$\$,IL8 and IL1$\$ response to challenge with bacterial endotoxin LPS,but lack response from genes associated with innate immune response in other cell types. Further,stimulation of cells with TNF-$\$ in auto-induction of TNF$\$,as well as cytokine responses of IL8 and IL1$\$ The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally,we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation. View Publication

Autosomal recessive osteopetrosis (OP) is a rare,lethal disorder in which osteoclasts are absent or nonfunctional,resulting in a bone marrow cavity insufficient to support hematopoiesis. Because osteoclasts are derived from hematopoietic precursors,allogeneic hematopoietic cell transplantation can cure the bony manifestations of the disorder. However,high rates of graft failure have been observed in this population. It is not possible to harvest bone marrow from these patients for reinfusion should graft failure be observed. We report that 8 of 10 patients with OP had high numbers of circulating CD34(+) cells (3% +/- 0.9%). This increased proportion of peripheral CD34(+) cells made it possible to harvest 2 x 10(6) CD34(+) cells per kilogram with a total volume of blood ranging from 8.3 to 83.7 mL (1.3-11.6 mL/kg). In addition,colony-forming assays documented significantly more colony-forming unit-granulocyte-macrophage and burst-forming unit-erythroid in the blood of osteopetrotic patients compared with controls; the numbers of colony-forming units approximated those found in control marrow. We conclude that OP patients with high levels of circulating CD34(+) are candidates for peripheral blood autologous harvest by limited exchange transfusion. These cells are then available for reinfusion should graft failure be observed in patients for whom retransplantation is impractical. View Publication

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC),we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea,Janus kinase 2 (JAK2) inhibitors,or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells,which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore,sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]),followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA),or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice,the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs,short-term and long-term SCID repopulating cells (SRCs),are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs,therefore,represents a possible effective means of treating PMF at the level of the malignant SRC. View Publication

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here,we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA,12.5% of cell colonies demonstrated CCR5 editing,of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells,we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells,including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication,macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro,and generation of HIV-resistant cells for potential therapeutic applications. View Publication

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression,which in turn is driven by promoters,enhancers,insulators and other cis-regulatory DNA sequences for each gene,the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters,enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast,enhancers are marked with highly cell-type-specific histone modification patterns,strongly correlate to cell-type-specific gene expression programs on a global scale,and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome,significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression. View Publication

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process,we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal,but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked,and they are available for researchers. View Publication

AIMS: Circular RNA (circRNA) is a newly validated class of single-stranded RNA,ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types,genome-wide circRNA expression in the heart has yet to be described in detail. METHODS AND RESULTS: We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts,25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools,we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse,respectively. Their abundance generally correlates with the abundance of their cognate linear RNA,but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN),RYR2,and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms,the majority (83%) of which originates from the I-band domain. Finally,we confirmed the expression of selected cardiac circRNA by RT-PCR,Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. CONCLUSIONS: Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA. View Publication

The family of calcium activated potassium channels of low and intermediate conductance,known as SK channels,consists of four members (SK1-4). These channels are widely expressed throughout the organism and involved in various cellular processes,such as the afterhyperpolarization in excitable cells but also in differentiation processes of various tissues. To date,the role of SK channels in developmental processes has been merely a marginal focus of investigation,although it is well accepted that cell differentiation and maturation affect the expression patterns of certain ion channels. Recently,several studies from our laboratory delineated the influence of SK channel expression and their respective activity on cytoskeletal reorganization in neural and pluripotent stem cells and regulation of cell fate determination toward the cardiac lineage in human and mouse pluripotent stem cells. Herein,we have now analyzed SK channel expression patterns and distribution at various stages of human induced pluripotent stem cell-derived neurogenesis particularly focusing on undifferentiated iPS cells,neural progenitors and mature neurons. All family members could be detected starting at the iPS cell level and were differentially expressed during the subsequent maturation process. Intriguingly,we found obvious discrepancies between mRNA and protein expression pointing toward a complex regulatory mechanism. Inhibition of SK channels with either apamin or clotrimazol did not have any significant effects on the speed or amount of neurogenesis in vitro. The abundance and specific regulation of SK channel expression during iPS cell differentiation indicates distinct roles of these ion channels not only for the cardiac but also for neuronal cell differentiation and in vitro neurogenesis. ?? 2013 Elsevier GmbH. View Publication

With defined culture protocol,human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro,therefore providing a great model for human heart development,and holding great potential for cardiac disease therapies. In this study,we successfully generated a highly pure population of human cardiomyocytes (hCMs) (backslashtextgreater95% cTnT+) from hESC line,which enabled us to identify and characterize an hCM-specific signature,at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation,and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover,we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression,providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types. View Publication

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Despite major progress in the management of human liver disease,the only cure for a critically failing organ is liver transplantation. While a highly successful approach,the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore,the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments,there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process,offering new treatment or preventative strategies. With both approaches in mind,we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly,our procedure is highly efficient (∼ 90%) and delivers active,drug-inducible,and predictive human hepatocyte populations. View Publication

Herpes simplex type 1 (HSV-1) amplicon vectors possess a number of features that make them excellent vectors for the delivery of transgenes into stem cells. HSV-1 amplicon vectors are capable of efficiently transducing both dividing and nondividing cells and since the virus is quite large,152 kb,it is of sufficient size to allow for incorporation of entire genomic DNA loci with native promoters. HSV-1 amplicon vectors can also be used to incorporate and deliver to cells a variety of sequences that allow extrachromosomal retention. These elements offer advantages over integrating vectors as they avoid transgene silencing and insertional mutagenesis. The construction of amplicon vectors carrying extrachromosomal retention elements,their packaging into HSV-1 viral particles,and the use of HSV-1 amplicons for stem cell transduction will be described. View Publication