技术资料

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication

Combinations of MAP/ERK kinase (MEK) and phosphoinositide 3-kinase (PI3K) inhibitors have shown promise in preclinical cancer models,leading to the initiation of clinical trials cotargeting these two key cancer signaling pathways. GDC-0973,a novel selective MEK inhibitor,and GDC-0941,a class I PI3K inhibitor,are in early stage clinical trials as both single agents and in combination. The discovery of these selective inhibitors has allowed investigation into the precise effects of combining inhibitors of two major signaling branches downstream of RAS. Here,we investigated multiple biomarkers in the mitogen-activated protein kinase (MAPK) and PI3K pathway to search for points of convergence that explain the increased apoptosis seen in combination. Using washout studies in vitro and alternate dosing schedules in mice,we showed that intermittent inhibition of the PI3K and MAPK pathway is sufficient for efficacy in BRAF and KRAS mutant cancer cells. The combination of GDC-0973 with the PI3K inhibitor GDC-0941 resulted in combination efficacy in vitro and in vivo via induction of biomarkers associated with apoptosis,including Bcl-2 family proapoptotic regulators. Therefore,these data suggest that continuous exposure of MEK and PI3K inhibitors in combination is not required for efficacy in preclinical cancer models and that sustained effects on downstream apoptosis biomarkers can be observed in response to intermittent dosing. View Publication

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application,but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification. We report a second harmonic generation (SHG) signal detectable in PSC-CMs that is attributable to sarcomeric myosin,dependent on PSC-CM maturity,and retained while PSC-CMs are in suspension. Our study demonstrates the feasibility of developing a SHG-activated flow cytometer for the non-invasive purification of PSC-CMs. View Publication

Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids,pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction,the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step,3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors,and they proliferate and expand over 1-3 months to give rise to intestinal tissue,complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date,this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro. View Publication

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication

Pluripotent stem cells are able to give rise to all cell types of the organism. There are two sources for human pluripotent stem cells: embryonic stem cells (ESCs) derived from surplus blastocysts created for in vitro fertilization and induced pluripotent stem cells (iPSCs) generated by reprogramming of somatic cells. ESCs have been an area of intense research during the past decade,and two clinical trials have been recently approved. iPSCs were created only recently,and most of the research has been focused on the iPSC generation protocols and investigation of mechanisms of direct reprogramming. The iPSC technology makes possible to derive pluripotent stem cells from any patient. However,there are a number of hurdles to be overcome before iPSCs will find a niche in practice. In this review,we discuss differences and similarities of the two pluripotent cell types and assess prospects for application of these cells in biomedicine. View Publication

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK),a key regulator for controlling energy metabolism,is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest,without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers,Nanog and stage-specific embryonic antigen-1,in the presence of leukemia inhibitory factor,without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently,an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation,an effect inhibited by MG132,a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs,it increased expression levels of erythroid cell lineage markers (Ter119,GATA1,Klf1,Hbb-b,and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR,endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs. View Publication

The accurate measurement of biologically active erythropoietin (Ep) in human serum and plasma using present in vivo and in vitro bioassays is difficult because of the presence of both inhibitors and non-Ep stimulators of erythropoiesis. We have developed a simple procedure to quantitatively purify Ep from serum and plasma for subsequent testing in the phenylhydrazine-treated mouse spleen cell assay. The method involves absorption of Ep to an immobilized high-affinity anti-Ep monoclonal antibody and acid elution of the antibody-bound material. After neutralization,the eluted EP is then tested directly in the in vitro bioassay without interference by other serum proteins. By using magnetic beads as a solid support for the antibody,washing and elution steps can be performed rapidly and efficiently. Recoveries of Ep after this procedure show very little sample-to-sample variation and are consistently between 45% and 55%,which is close to the maximum binding expected for the anti-Ep antibody. Coupled with the 7.4-fold concentration that this procedure affords,there is an overall increase in sensitivity of three- to fourfold,which makes this assay suitable for accurately measuring Ep levels in patients with below-average titers. Results with this magnetic bead assay indicate that accurate and reproducible estimates for Ep levels in the serum and plasma from healthy donors as well as from patients with hematologic disorders can be obtained. Titers of biologically active Ep in the sera from a group of patients with either leukemia or lymphoma were found to be elevated,and the values correlated well with titers of immunoreactive Ep measured in the Ep radioimmunoassay. Because of its specificity and high sensitivity,the magnetic bead assay is a valuable alternative to immunoassays for the measurement of elevated,normal,and even subnormal Ep levels in human serum and plasma. View Publication

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here,we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells,as predicted from a numerical model of cell migration,and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further,reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications. View Publication

Metabolism is vital to every aspect of cell function,yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report,using an untargeted metabolomics approach,that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells,and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis,and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly,the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs,which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs,and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency,and in evaluating iPSC and ESC equivalence. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation,and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI,and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I),we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI,we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation,and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together,our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation,and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. View Publication