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The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines,from 38 laboratories worldwide,for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal,but there was a progressive tendency to acquire changes on prolonged culture,commonly affecting chromosomes 1,12,17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants,determined from the SNP arrays,also appeared sporadically. No common variants related to culture were observed on chromosomes 1,12 and 17,but a minimal amplicon in chromosome 20q11.21,including three genes expressed in human ES cells,ID1,BCL2L1 and HM13,occurred in textgreater20% of the lines. Of these genes,BCL2L1 is a strong candidate for driving culture adaptation of ES cells. View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes,yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified,homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here,we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized,and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding,homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103 View Publication

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However,the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs,we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast,overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover,TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways,reversing drug resistance and inhibiting the metastasis of cancer cells. View Publication

Chondrogenesis of mesenchymal stem cells (MSCs) is typically induced when they are condensed into a single aggregate and exposed to transforming growth factor-beta (TGF-beta). Hypoxia,like aggregation and TGF-beta delivery,may be crucial for complete chondrogenesis. However,the pellet dimensions and associated self-induced oxygen gradients of current chondrogenic methods may limit the effectiveness of in vitro differentiation and subsequent therapeutic uses. Here we describe the use of embryoid body-forming technology to produce microscopic aggregates of human bone marrow MSCs (BM-MSCs) for chondrogenesis. The use of micropellets reduces the formation of gradients within the aggregates,resulting in a more homogeneous and controlled microenvironment. These micropellet cultures (approximately 170 cells/micropellet) as well as conventional pellet cultures (approximately 2 x 10(5) cells/pellet) were chondrogenically induced under 20% and 2% oxygen environments for 14 days. Compared to conventional pellets under both environments,micropellets differentiated under 2% O(2) showed significantly increased sulfated glycosaminoglycan (sGAG) production and more homogeneous distribution of proteoglycans and collagen II. Aggrecan and collagen II gene expressions were increased in pellet cultures differentiated under 2% O(2) relative to 20% O(2) pellets but 2% O(2) micropellets showed even greater increases in these genes,as well as increased SOX9. These results suggest a more advanced stage of chondrogenesis in the micropellets accompanied by more homogeneous differentiation. Thus,we present a new method for enhancing MSC chondrogenesis that reveals a unique relationship between oxygen tension and aggregate size. The inherent advantages of chondrogenic micropellets over a single macroscopic aggregate should allow for easy integration with a variety of cartilage engineering strategies. View Publication

Pluripotency,the ability of a cell to differentiate and give rise to all embryonic lineages,defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes,accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells,as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency,we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation,except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression,suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers,and observed much greater dynamics in chromatin modifications,especially H3K4me1 and H3K27ac,which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells,chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG,SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture,greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9,type II collagen,and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen,type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state,suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects,and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs. ?? 2014 Elsevier Ltd. View Publication

Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here,we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable,and endothelial progenitor differentiation was β-catenin dependent. Furthermore,by clonal analysis,we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent,capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings,formed tube-like structures,imported acetylated low-density lipoprotein,and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/β-catenin signaling as a primary regulator for generating vascular cells from hPSCs. View Publication

WNT/$\$-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3$\$ (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]),we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes,which are expressed in the posterior mesoderm of developing embryos,were significantly upregulated by GSKi treatment. Further,inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs,and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore,transient WNT/$\$-CATENIN signaling triggers activation of the CDX/HOX pathway,which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs. View Publication