技术资料

The development of xeno-free,chemically defined stem cell culture systems has been a primary focus in the field of regenerative medicine to enhance the clinical application of pluripotent stem cells (PSCs). In this regard,various electrospun substrates with diverse physiochemical properties were synthesized utilizing various polymer precursors and surface treatments. Human induced pluripotent stem cells (IPSCs) cultured on these substrates were characterized by their gene and protein expression to determine the effects of the substrate physiochemical properties on the cells' self-renewal,i.e.,proliferation and the maintenance of pluripotency. The results showed that surface chemistry significantly affected cell colony formation via governing the colony edge propagation. More importantly,when surface chemistry of the substrates was uniformly controlled by collagen conjugation,the stiffness of substrate was inversely related to the sphericity,a degree of three dimensionality in colony morphology. The differences in sphericity subsequently affected spontaneous differentiation of IPSCs during a long-term culture,implicating that the colony morphology is a deciding factor in the lineage commitment of PSCs. Overall,we show that the capability of controlling IPSC colony morphology by electrospun substrates provides a means to modulate IPSC self-renewal. View Publication

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein,tandem dimer Tomato (tdTomato),are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons,evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations. View Publication

BACKGROUND: Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body. Ets variant 2 (ETV2) is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification. Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells. Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.backslashnbackslashnFINDINGS: We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells (ESCs) to determine when the peak of ETV2 expression occurs. We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.backslashnbackslashnCONCLUSIONS: Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification. This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic. View Publication

The generation of personalized induced pluripotent stem cells (iPSCs) followed by targeted genome editing provides an opportunity for developing customized effective cellular therapies for genetic disorders. However,it is critical to ascertain whether edited iPSCs harbor unfavorable genomic variations before their clinical application. To examine the mutation status of the edited iPSC genome and trace the origin of possible mutations at different steps,we have generated virus-free iPSCs from amniotic cells carrying homozygous point mutations in beta-hemoglobin gene (HBB) that cause severe beta-thalassemia (beta-Thal),corrected the mutations in both HBB alleles by zinc finger nuclease-aided gene targeting,and obtained the final HBB gene-corrected iPSCs by excising the exogenous drug resistance gene with Cre recombinase. Through comparative genomic hybridization and whole-exome sequencing,we uncovered seven copy number variations,five small insertions/deletions,and 64 single nucleotide variations (SNVs) in beta-Thal iPSCs before the gene targeting step and found a single small copy number variation,19 insertions/deletions,and 340 single nucleotide variations in the final gene-corrected beta-Thal iPSCs. Our data revealed that substantial but different genomic variations occurred at factor-induced somatic cell reprogramming and zinc finger nuclease-aided gene targeting steps,suggesting that stringent genomic monitoring and selection are needed both at the time of iPSC derivation and after gene targeting. View Publication

Nature Genetics 47,469 (2015). doi:10.1038/ng.3258 View Publication

For diseases of the brain,the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus,POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro,retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages,and responded to retinoic acid,promoting a more posterior fate (HOXB4+,OTX2-,and GBX2-). These findings offer insight into pig iPSC development,which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models,providing relevant translational data for eventual human neural cell therapies. View Publication

Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults,respectively. In both of these cases,a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue,wound healing complications,and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need,we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures,degradation rates,and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro,silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling. View Publication

In recent years,several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here,we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore,we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly,these genes are also associated with skin disorders and ectodermal defects,providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions. View Publication

Environmental influences and insults by reproductive toxicant exposure can lead to impaired spermatogenesis or infertility. Understanding how toxicants disrupt spermatogenesis is critical for determining how environmental factors contribute to impaired fertility. While current animal models are available,understanding of the reproductive toxic effects on human fertility requires a more robust model system. We recently demonstrated that human pluripotent stem cells can differentiate into spermatogonial stem cells/spermatogonia,primary and secondary spermatocytes,and haploid spermatids; a model that mimics many aspects of human spermatogenesis. Here,using this model system,we examine the effects of 2-bromopropane (2-BP) and 1,2,dibromo-3-chloropropane (DBCP) on in vitro human spermatogenesis. 2-BP and DBCP are non-endocrine disrupting toxicants that are known to impact male fertility. We show that acute treatment with either 2-BP or DBCP induces a reduction in germ cell viability through apoptosis. 2-BP and DBCP affect viability of different cell populations as 2-BP primarily reduces spermatocyte viability,whereas DBCP exerts a much greater effect on spermatogonia. Acute treatment with 2-BP or DBCP also reduces the percentage of haploid spermatids. Both 2-BP and DBCP induce reactive oxygen species (ROS) formation leading to an oxidized cellular environment. Taken together,these results suggest that acute exposure with 2-BP or DBCP causes human germ cell death in vitro by inducing ROS formation. This system represents a unique platform for assessing human reproductive toxicity potential of various environmental toxicants in a rapid,efficient,and unbiased format. View Publication

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently,chromatin has been shown to play a part in this process. To elucidate this relationship further,we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed,that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally,cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts,and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed,our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. View Publication

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical,environmental,and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part,previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation,which are highly nonphysiologic,or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair,we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs,compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus,the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny. View Publication

Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general,but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies,however,is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is,however,limited and thereby further optimization in terms of time,efficiency,and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts,at least in part,in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1,Inhba and Grem1. Hence,we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. View Publication