技术资料

The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover,these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology,positive staining for alkaline phosphatase,normal karyotype,and expression of hESC-like markers including Nanog,Rex1,Oct4,TRA-1-60,TRA-1-80,SSEA-3,and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers,and could as well be readily differentiated into functional motor neurons. Among other things,our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming,differentiation ability,stability of the resulting lineages,and the risk of associated abnormalities. View Publication

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage,high histologic grade,recurrence and progression,and metastasis of bladder urothelial cell carcinomas (all P textless 0.05). Furthermore,ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030,respectively). Therefore,ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments. View Publication

This work describes a genetic approach to isolate small,artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric,44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small,dimeric TM proteins that were structurally unrelated to erythropoietin (EPO),but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore,this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus,we have changed the specificity of a protein so that it no longer recognizes its natural target but,instead,modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word traptamer" for these transmembrane aptamers." View Publication

Adipogenesis plays a key role in the pathogenesis of obesity. It begins with the commitment of mesenchymal stem cells (MSCs) to the adipocyte lineage,followed by terminal differentiation of preadipocytes to mature adipocytes. A critical,but poorly understood,component of adipogenesis involves proliferation of MSCs and preadipocytes. The present study was undertaken to examine the hypothesis that bone morphogenetic protein-3 (BMP-3) promotes adipogenesis using C3H10T1/2 MSCs and 3T3-L1 preadipocytes as in vitro model systems. We demonstrated that although it did not promote the commitment of MSCs to the adipocyte lineage or the differentiation of preadipocytes to adipocytes,BMP-3-stimulated proliferation by threefold in both cell types. Owing to a lack of information on MSC proliferation,we then delineated the molecular mechanisms underlying BMP-3-stimulated MSC proliferation. We showed that BMP-3 activated the transforming growth factor-beta (TGF-beta)/activin but not ERK1/2,p38 MAPK,or JNK signaling pathways in C3H10T1/2 cells. Furthermore,the TGF-beta/activin receptor kinase inhibitor SB-431542 blocked BMP-3-stimulated proliferation. Importantly,siRNA-mediated knockdown of the key TGF-beta/activin signaling pathway components,ActRIIB,ALK4,or Smad2,abrogated the mitogenic effects of BMP-3 on MSCs. Together,these results demonstrate that BMP-3 stimulates MSC proliferation via the TGF-beta/activin signaling pathway,thus revealing a novel role for this divergent and poorly understood member of the TGF-beta superfamily in regulating MSC proliferation. View Publication

NOTCH signaling is critical for specifying the intestinal epithelial cell lineage and for initiating colorectal adenomas and colorectal cancers (CRC). Based on evidence that NOTCH is important for the maintenance and self-renewal of cancer-initiating cells in other malignancies,we studied the role of NOTCH signaling in colon cancer-initiating cells (CCIC). Tumors formed by CCICs maintain many properties of the primary CRCs from which they were derived,such as glandular organization,cell polarity,gap junctions,and expression of characteristic CRC molecular markers. Furthermore,CCICs have the property of self-renewal. In this study,we show that NOTCH signaling is 10- to 30-fold higher in CCIC compared with widely used colon cancer cell lines. Using small-molecule inhibition and short hairpin RNA knockdown,we show that NOTCH prevents CCIC apoptosis through repression of cell cycle kinase inhibitor p27 and transcription factor ATOH1. NOTCH is also critical to intrinsic maintenance of CCIC self-renewal and the repression of secretory cell lineage differentiation genes such as MUC2. Our findings describe a novel human cell system to study NOTCH signaling in CRC tumor initiation and suggest that inhibition of NOTCH signaling may improve CRC chemoprevention and chemotherapy. View Publication

Increased proportions of CD8 T lymphocytes lacking expression of the CD28 costimulatory receptor have been documented during both aging and chronic infection with HIV-1,and their abundance correlates with numerous deleterious clinical outcomes. CD28-negative cells also arise in cell cultures of CD8(+)CD28(+) following multiple rounds of Ag-driven proliferation,reaching the end stage of replicative senescence. The present study investigates the role of a second T cell costimulatory receptor component,adenosine deaminase (ADA),on the process of replicative senescence. We had previously reported that CD28 signaling is required for optimal telomerase upregulation. In this study,we show that the CD8(+)CD28(+) T lymphocytes that are ADA(+) have significantly greater telomerase activity than those that do not express ADA and that ADA is progressively lost as cultures progress to senescence. Because ADA converts adenosine to inosine,cells lacking this enzyme might be subject to prolonged exposure to adenosine,which has immunosuppressive effects. Indeed,we show that chronic exposure of CD8 T lymphocytes to exogenous adenosine accelerates the process of replicative senescence,causing a reduction in overall proliferative potential,reduced telomerase activity,and blunted IL-2 gene transcription. The loss of CD28 expression was accelerated,in part due to adenosine-induced increases in constitutive caspase-3,known to act on the CD28 promoter. These findings provide the first evidence for a role of ADA in modulating the process of replicative senescence and suggest that strategies to enhance this enzyme may lead to novel therapeutic approaches for pathologies associated with increases in senescent CD8 T lymphocytes. View Publication

The use of human pluripotent stem cells,including embryonic and induced pluripotent stem cells,in therapeutic applications will require the development of robust,scalable culture technologies for undifferentiated cells. Advances made in large-scale cultures of other mammalian cells will facilitate expansion of undifferentiated human embryonic stem cells (hESCs),but challenges specific to hESCs will also have to be addressed,including development of defined,humanized culture media and substrates,monitoring spontaneous differentiation and heterogeneity in the cultures,and maintaining karyotypic integrity in the cells. This review will describe our current understanding of environmental factors that regulate hESC self-renewal and efforts to provide these cues in various scalable bioreactor culture systems. View Publication

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine,levels of which are modulated by stress,depression and clinical antidepressants. However,studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors,including stem cells,from the hippocampus of adult mice. Mechanistically,we provide evidence that beta(3)-adrenergic receptors,which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ),mediate this norepinephrine-dependent activation. Moreover,intrahippocampal injection of a selective beta(3)-adrenergic receptor agonist in vivo increases the number of proliferating cells in the SGZ. Similarly,systemic injection of the beta-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursors in vivo. Finally,using a novel ex vivo slice-sphere" assay that maintains an intact neurogenic niche� View Publication

Patients with dyskeratosis congenita (DC),a disorder of telomere maintenance,suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity,which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state,that several telomerase components are targeted by pluripotency-associated transcription factors,and that in autosomal dominant DC,transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase,and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients. View Publication

A series of chalcones (1-9) and pyrazoles (10-18) was prepared to investigate their potential activity as Angiotensin I-Converting Enzyme (ACE) inhibitors. Their structures were verified by elemental analysis,UV,IR,MS,(1)H NMR,(13)C NMR,and 2D NMR experiments. Among tested compounds,chalcone 7 exerted the highest activity with an IC(50) value of 0.219 mM,while the most potent pyrazole was 15 (IC(50) value of 0.213 mM). View Publication

A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics,most important of which is the pluripotency,hESC lines vary significantly in their transcriptional profiles,genetic,and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences,the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report,we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria,including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines,namely hESM01-04,were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free,serum-free conditions using mTeSR1 and Matrigel. The fifth line,hESMK05 was derived in feeder-free,serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community. View Publication

We had earlier reported the derivation and characterization of two new sibling human embryonic stem cell lines BJNhem19 and BJNhem20,from discarded grade III embryos of Indian origin. We report here the characteristics of the two sibling cell lines after long-term continuous culture for over 2 yr during which they have been passaged over 200 times. We show that both cell lines adapt well to culture on various mouse and human feeders as well as in feeder-free conditions. The cells show normal diploid karyotype and continue to express all pluripotency markers. Both cell lines differentiate to derivatives of all three germ layers in vitro. However as reported earlier,BJNhem19 is unable to generate teratomas in nude or SCID mice or differentiate to beating cardiomyocytes when tested over several passages during long-term stable culture. On the other hand,the cardiac differentiation capacity of BJNhem20 is greatly increased,and it can generate beating cardiomyocytes that proliferate when isolated and cultured further. In conclusion,the two cell lines have maintained a stable phenotype for over 2 yr and are indeed immortal. Their derivation from grade III embryos does not seem to have any adverse effect on their long-term phenotype. The cells can be obtained for research purposes from the UK Stem Cell Bank and from the authors. View Publication