技术资料

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product,the amyloid-beta (Abeta) protein,is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease,Down syndrome,and head injury. We recently reported that this protein,normally associated with neurodegenerative conditions,is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta,which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression,an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha,which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis. View Publication

Dax-1 (Nr0b1) is an orphan member of the nuclear hormone receptor superfamily that has a key role in adrenogonadal development and function. Recent studies have also implicated Dax-1 in the transcriptional network controlling embryonic stem (ES) cell pluripotency. Here,we show that Dax-1 expression is affected by differentiating treatments and pharmacological activation of beta-catenin-dependent transcription in mouse ES cells. Furthermore,Dax-1 knockdown induced upregulation of multilineage differentiation markers,and produced enhanced differentiation and defects in ES viability and proliferation. Through RNA interference and transcriptome analysis,we have identified genes regulated by Dax-1 in mouse ES cells at 24 and 48 hours after knockdown. Strikingly,the great majority of these genes are upregulated,showing that the prevalent function of Dax-1 is to act as a transcriptional repressor in mouse ES cells,as confirmed by experiments using the Gal4 system. Genes involved in tissue differentiation and control of proliferation are significantly enriched among Dax-1-regulated transcripts. These data show that Dax-1 is an essential element in the molecular circuit involved in the maintenance of ES cell pluripotency and have implications for the understanding of stem cell function in both physiological (adrenal gland) and clinical (Ewing tumors) settings where Dax-1 plays a pivotal role in development and pathogenesis,respectively. View Publication

The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins,progesterone (P(4)),human chorionic gonadotropin,17beta-estradiol,and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis,we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864,a delta-opioid receptor antagonist,and RU-486 (mifepristone),a P(4) receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies,an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore,these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression,an early marker of neural precursor cells normally detected during rosette formation. Conversely,addition of P(4) to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis. View Publication

Human embryonic stem cells hold great promise in furthering our treatment of disease and increasing our understanding of early development. This chapter describes protocols for the derivation and maintenance of human embryonic stem cells. In addition,it summarizes briefly several alternative methods for the culture of human embryonic stem cells. Thus,this chapter provides a good starting point for researchers interested in harnessing the potential of human embryonic stem cells. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. View Publication

Mechanisms of lymphoid and myeloid lineage choice by hemopoietic stem cells remain unclear. In this study we show that the multipotent progenitor (MPP) population,which is immediately downstream of hemopoietic stem cells,is heterogeneous and can be subdivided in terms of VCAM-1 expression. VCAM-1(+) MPPs were fully capable of differentiating into both lymphoid and myeloid lineages. In contrast,VCAM-1(-) MPPs gave rise to lymphocytes predominately in vivo. T and B cell development from VCAM-1(-) MPPs was 1 wk faster than that from VCAM-1(+) MPPs. Furthermore,VCAM-1(+) MPPs gave rise to common myeloid progenitors and VCAM-1(-) MPPs in vivo,indicating that VCAM-1(-) MPPs are progenies of VCAM-1(+) MPPs. VCAM-1(-) MPPs,in turn,developed into lymphoid lineage-restricted common lymphoid progenitors. These results establish a hierarchy of developmental relationship between MPP subsets and lymphoid and myeloid progenitors. In addition,VCAM-1(+) MPPs may represent the branching point between the lymphoid and myeloid lineages. View Publication

We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells,but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions. View Publication

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. View Publication

Definitive mesoderm arises from a bipotent mesendodermal population,and to study processes controlling its development at this stage,embryonic stem (ES) cells can be employed. SHB (Src homology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context,we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively,EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation,EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic,vascular,and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition,the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context. View Publication

Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently,murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4,Sox2,Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors,we have derived iPS cells from fetal,neonatal and adult human primary cells,including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency,and establish a method whereby patient-specific cells might be established in culture. View Publication

Defined growth conditions are essential for many applications of human embryonic stem cells (hESC). Most defined media are presently used in combination with Matrigel,a partially defined extracellular matrix (ECM) extract from mouse sarcoma. Here,we defined ECM requirements of hESC by analyzing integrin expression and ECM production and determined integrin function using blocking antibodies. hESC expressed all major ECM proteins and corresponding integrins. We then systematically replaced Matrigel with defined medium supplements and ECM proteins. Cells attached efficiently to natural human vitronectin,fibronectin,and Matrigel but poorly to laminin + entactin and collagen IV. Integrin-blocking antibodies demonstrated that alphaVbeta5 integrins mediated adhesion to vitronectin,alpha5beta1 mediated adhesion to fibronectin,and alpha6beta1 mediated adhesion to laminin + entactin. Fibronectin in feeder cell-conditioned medium partially supported growth on all natural matrices,but in defined,nonconditioned medium only Matrigel or (natural and recombinant) vitronectin was effective. Recombinant vitronectin was the only defined functional alternative to Matrigel,supporting sustained self-renewal and pluripotency in three independent hESC lines. View Publication