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Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling,axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development,the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks,gene expression and unique peak profiles associated with dysregulated genes,suggesting a coordinated epigenetic program. Treatment with isoxazole-9,which targets key dysregulated pathways,led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. View Publication

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity,and prepared them in formations of single chain,single chain with joint segment,dual chain with joint segment,and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain,which has a storage modulus of 25 kPa,supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. View Publication

In the field of hematopoietic regeneration,deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells,remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines,UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here,using a serum-free,feeder-free,and chemically defined induction protocol,we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34(+)CD45(+) derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol,the derived hematopoietic progenitors failed to engraft in NOG mice,indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols,UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro. View Publication

Monitoring pluripotent stem cell behaviors (self-renewal and differentiation to specific lineages/phenotypes) is critical for a fundamental understanding of stem cell biology and their translational applications. In this study,a multi-modal stem cell monitoring system was developed to quantitatively characterize physico-electrochemical changes of the cells in real time,in relation to cellular activities during self-renewal or lineage-specific differentiation,in a non-destructive,label-free manner. The system was validated by measuring physical (mass) and electrochemical (impedance) changes in human induced pluripotent stem cells undergoing self-renewal,or subjected to mesendodermal or ectodermal differentiation,and correlating them to morphological (size,shape) and biochemical changes (gene/protein expression). An equivalent circuit model was used to further dissect the electrochemical (resistive and capacitive) contributions of distinctive cellular features. Overall,the combination of the physico-electrochemical measurements and electrical circuit modeling collectively offers a means to longitudinally quantify the states of stem cell self-renewal and differentiation. View Publication

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair,underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here,we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines,including a WT1-2A-eGFP knock-in reporter line,under chemically-defined,xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells,resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo,as determined by morphological and functional assays,including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies. View Publication

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb,Gfi1,Runx1,and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells,which results in endogenous Runx1 expression. During the specification phase (days 8-20),RUNX1(+) FGRS-transduced endothelial cells commit to a haematopoietic fate,yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells,and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution,including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. View Publication

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens,or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here,to yield functional human haematopoietic stem cells,we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG,HOXA5,HOXA9,HOXA10,LCOR,RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid,B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. View Publication

Microglia,the immune cells of the brain,are crucial to proper development and maintenance of the CNS,and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology,we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes,expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca(2+) transients,whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines. View Publication

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed,iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro,phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation,validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data,typically by relating the obtained levels of gene expression to the levels of expression of a house keeping gene"� View Publication

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells,it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line). We examined three parameters related to cellular bioenergetics: phosphotransfer system,aerobic glycolysis,and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells. 2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85±5.73 vs 64.39±2.55mU/mg of protein) and the expression of AK2 was significantly higher in these cells,while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass,increased proton leak,and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference,and highlight the importance of further research concerning energy metabolism of stem cells. View Publication

Normal murine bone marrow (BM) cells were sorted on the basis of low forward and orthogonal light scatter properties,Sca-1 expression (Sca-1+),lack of staining with a cocktail of mature hematopoietic lineage markers (Lin-),and binding of wheat germ agglutinin (WGA+). This approach allowed the reproducible isolation of a very small subpopulation (0.037% +/- 0.023% of all nucleated BM cells) that was approximately 400-fold enriched in cells capable of reconstituting both lymphoid and myeloid lineages in lethally irradiated recipients. Transplantation of 30 or 10 of these Sca-1+Lin-WGA+ cells resulted in textgreater or = to 20% donor-derived nucleated peripheral blood cells 3 months posttransplantation in 100% and 22% of the recipients,respectively. When Sca-1+Lin-WGA+ cells were cultured in serum-free medium supplemented with Steel factor,interleukin-6 (IL-6),and erythropoietin (with or without IL-3),a large increase in total cell number,including cells with an Sca-1+Lin-WGA+ phenotype was observed. Single cell cultures showed that 90% to 95% of the input cells underwent at least one division during the first 2 weeks and the remainder died. Interestingly,this proliferative response was not accompanied by a parallel increase in the number of cells with both lymphoid and myeloid repopulating potential in vivo,as quantitation of these by limiting dilution analysis showed they had decreased slightly (1.3-fold) but not significantly below the number initially present. These results demonstrate that Sca-1+Lin-WGA+ cells with long-term repopulating potential can be maintained for 2 weeks in a serum- and stroma cell-free culture,providing a simple in vitro system to study their behavior under well-defined conditions. The observed expansion of Sca-1+Lin-WGA+ cells in vitro without a concomitant increase in reconstituting cells also shows that extensive functional heterogeneity exists within populations of cells with this surface phenotype. View Publication

Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit,glutamate receptor subunits,the transcription factor Brn-3,and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels,as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation. View Publication