技术资料

The generation of human induced pluripotent stem cells (hiPSC) from somatic cells has enabled the possibility to provide patient-specific hiPSC for cell-based therapy,drug discovery,and other translational applications. Two major obstacles in using hiPSC for clinical application reside in the risk of genomic modification when they are derived with viral transgenes and risk of teratoma formation if undifferentiated cells are engrafted. In this study,we report the generation of footprint-free" hiPSC-derived astrocytes. These are efficiently generated� View Publication

Native porcine nucleus pulposus (NP) tissue harbors a number of notochordal cells (NCs). Whether the native NP matrix supports the homeostasis of notochordal cells is poorly understood. We hypothesized the NP matrix alone may contain sufficient regulatory factors and can serve as stimuli to generate notochordal cells (NCs) from human pluripotent stem cells. NCs are a promising cell sources for cell-based therapy to treat some types of intervertebral disc (IVD) degeneration. One major limitation of this emerging technique is the lack of available NCs as a potential therapeutic cell source. Human pluripotent stem cells derived from reprogramming or somatic cell nuclear transfer technique may yield stable and unlimited source for therapeutic use. We devised a new method to use porcine NP matrix to direct notochordal differentiation of human induced pluripotent stem cells (hiPSCs). The results showed that hiPSCs successfully differentiated into NC-like cells under the influence of devitalized porcine NP matrix. The NC-like cells expressed typical notochordal marker genes including brachyury (T),cytokeratin-8 (CK-8) and cytokeratin-18 (CK-18),and they displayed the ability to generate NP-like tissue in vitro,which was rich in aggrecan and collagen type II. These findings demonstrated the proof of concept for using native NP matrix to direct notochordal differentiation of hiPSCs. It provides a foundation for further understanding the biology of NCs,and eventually towards regenerative therapies for disc degeneration. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells and human-induced pluripotent stem cells,are a renewable cell source for a wide range of applications in regenerative medicine and useful tools for human disease modeling and drug discovery. For these purposes,large numbers of high-quality cells are essential. Recently,we showed that a biological substrate,recombinant E8 fragments of laminin isoforms,sustains long-term self-renewal of hPSCs in defined,xeno-free medium with dissociated single-cell passaging. Here,we describe a modified culture system with similar performance to efficiently expand hPSCs under defined,xeno-free conditions using a non-biological synthetic substrate. View Publication

Human induced pluripotent stem cells (hiPSCs),like embryonic stem cells,are under intense investigation for novel approaches to model disease and for regenerative therapies. Here,we describe the derivation and characterization of hiPSCs from a variety of sources and show that,irrespective of origin or method of reprogramming,hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium,CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage,we were able to demonstrate by single cell analysis (flow cytometry),that hiPSC-derived erythroblasts express alpha globin as previously described,and that a sub-population of these erythroblasts also express haemoglobin F (HbF),indicative of fetal definitive erythropoiesis. More notably however,we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner,but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover,the HbA expressing erythroblast population could be greatly enhanced (44textperiodcentered0 ± 6textperiodcentered04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years,evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review,we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition,we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium. View Publication

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC,these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further,the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial,ventricular (V),and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2,H7,and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4,Rho kinase inhibitor,activin-A,and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16,up to 95% of cells were cTnT(+). Of which,93%,94%,100%,92%,and 92% of cardiac derivatives from HES2,H7,H9,and two iPSC lines,respectively,were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to $\$-adrenergic stimulation. Our simple,cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol,the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors. View Publication

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics,but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge,we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs),pericytes,astrocytes and neurons derived from renewable cell sources. First,retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs,particularly through adherens junction,tight junction,and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (˜5,000 $\$(2)). Overall,this scalable human BBB model may enable a wide range of neuroscience studies. View Publication

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study,we tested whether functional differences between chronic obstructive pulmonary disease (COPD) and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4,nanog,and sox2,formed embryoid bodies in vitro,and induced teratomas in nonobese diabetic/severe combined immunodeficient mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared with their corresponding primary fibroblasts. Primary COPD fibroblasts contracted three-dimensional collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast,redifferentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1,881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts,with 605 genes differing by more than twofold. After redifferentiation,112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than twofold. Similar findings were observed with microRNA (miRNA) expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation,only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly,of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts,293 genes were changed toward control after redifferentiation. In conclusion,functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs. View Publication

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence,protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation. The integrity of cell:cell contacts has been shown to influence lineage choice during pluripotent cell differentiation,with disruption of cell:cell contacts promoting mesendoderm formation and maintenance of cell:cell contacts resulting in the preferential formation of neurectoderm. Staurosporine is a broad spectrum inhibitor of serine/threonine kinases which has several effects on cell function,including interruption of cell:cell contacts,decreasing focal contact size,inducing epithelial to mesenchyme transition (EMT) and promoting cell differentiation. The possibility that staurosporine could influence lineage choice from pluripotent cells in culture was investigated. The addition of staurosporine to differentiating mouse EPL resulted in preferential formation of mesendoderm and mesoderm populations,and inhibited the formation of neurectoderm. Addition of staurosporine to human ES cells similarly induced primitive streak marker gene expression. These data demonstrate the ability of staurosporine to influence lineage choice during pluripotent cell differentiation and to mimic the effect of disrupting cell:cell contacts. Staurosporine induced mesendoderm in the absence of known inducers of formation,such as serum and BMP4. Staurosporine induced the expression of mesendoderm markers,including markers that were not induced by BMP4,suggesting it acted as a broad spectrum inducer of molecular gastrulation. This approach has identified a small molecule regulator of lineage choice with potential applications in the commercial development of ES cell derivatives,specifically as a method for forming mesendoderm progenitors or as a culture adjunct to prevent the formation of ectoderm progenitors during pluripotent cell differentiation. ?? 2014. View Publication