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Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis,and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here,we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus,a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus,this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome. View Publication

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts,perceptions,and emotions,usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however,most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells,derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient,presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS),when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia,contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening. View Publication

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

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan,a 61-amino acid vasodilatory peptide,specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells,little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study,we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1,we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC,the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells,as demonstrated by WST-8 analysis,annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly,immunofluorescence,western blot analysis,real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover,karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary,these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. View Publication

Despite major advances in the pathophysiological understanding of peripheral nerve damage,the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end,we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival,migration,and guide neurite extension. View Publication

Pluripotent stem cells,including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs),have the potential to treat type 1 diabetes through cell replacement therapy. However,the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification,and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation,and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified,suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR,and the relationship between one of these miRNAs,miR-151a-5p,and its predicted target gene,SOX17,was investigated by luciferase assay,and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion,these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells. View Publication

Reliance on volunteer blood donors can lead to transfusion product shortages,and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time,known as 'the storage lesion'. Thus,there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh,transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However,it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity,viability,deformability,and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo,we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel,chronically anemic,SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data,stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs,the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product. View Publication

BACKGROUND: The histone variant H2A.Z has been implicated in nucleosome exchange,transcriptional activation and Polycomb repression. However,the relationships among these seemingly disparate functions remain obscure.backslashnbackslashnRESULTS: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers,and correlates strongly with H3K4 methylation. Accordingly,H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation,but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z,including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.backslashnbackslashnCONCLUSIONS: Our findings associate H2A.Z with functionally distinct genomic elements,and suggest that post-translational modifications may reconcile its contrasting locations and roles. 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

Many studies have compared the genetic and epigenetic profiles of human induced pluripotent stem cells (hiPSCs) to human embryonic stem cells (hESCs) and yet the picture remains unclear. To address this,we derived a population of neural precursor cells (NPCs) from the H1 (WA01) hESC line and generated isogenic iPSC lines by reprogramming. The gene expression and methylation profile of three lines were compared to the parental line and intermediate NPC population. We found no gene probe with expression that differed significantly between hESC and iPSC samples under undifferentiated or differentiated conditions. Analysis of the global methylation pattern also showed no significant difference between the two PSC populations. Both undifferentiated populations were distinctly different from the intermediate NPC population in both gene expression and methylation profiles. One point to note is that H1 is a male line and so extrapolation to female lines should be cautioned. However,these data confirm our previous findings that there are no significant differences between hESCs and hiPSCs at the gene expression or methylation level. View Publication

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation,we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling,paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types,indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus,iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover,altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations. View Publication