技术资料

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

BACKGROUND: Tissue damage after hematopoietic stem cell transplantation (HSCT) occurs as a result of high-dose chemotherapy and radiation. The aim was to determine the importance of pretransplant anemia on toxicity and red blood cell (RBC) transfusion requirements after autologous HSCT. STUDY DESIGN AND METHODS: A total of 350 patients undergoing autologous HSCT were included in the analysis. Patient factors and pretransplant laboratory values of possible relevance were assessed in multivariate regression analysis. RESULTS: Reduced hemoglobin (Hb) on the first day of peripheral blood progenitor cell (PBPC) collection was significantly associated with increased organ toxicity after HSCT,as measured by the Seattle criteria. Lower Hb levels at baseline before transplantation,but not at PBPC collection,were significantly associated with increased RBC transfusion requirements. In a second cohort of 28 patients,higher Hb levels on the day of PBPC collection were significantly associated with increased levels of endothelial-like vascular progenitor cells in PBPC grafts. CONCLUSION: Our observations suggest that higher Hb levels on the day of PBPC collection may be a marker of reduced toxicity associated with HSCT and increased vascular progenitors in PBPC collections. Further,baseline anemia before transplant may reflect an unfavorable hematopoietic microenvironment that leads to increased RBC transfusion requirements. View Publication

BACKGROUND: Embryonic stem (ES) cells self-renew as coherent colonies in which cells maintain tight cell-cell contact. Although intercellular communications are essential to establish the basis of cell-specific identity,molecular mechanisms underlying intrinsic cell-cell interactions in ES cells at the signaling level remain underexplored.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: Here we show that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ES cells. siRNA-mediated loss of function experiments demonstrated that Rock,a major effector kinase downstream of Rho,played a key role in the formation of cell-cell junctional assemblies through regulation of myosin II by controlling a myosin light chain phosphatase. Chemical engineering of this signaling axis by a Rock-specific inhibitor revealed that cell-cell adhesion was reversibly controllable and dispensable for self-renewal of mouse ES cells as confirmed by chimera assay. Furthermore,a novel culture system combining a single synthetic matrix,defined medium,and the Rock inhibitor fully warranted human ES cell self-renewal independent of animal-derived matrices,tight cell contacts,or fibroblastic niche-forming cells as determined by teratoma formation assay.backslashnbackslashnCONCLUSIONS/SIGNIFICANCE: These findings demonstrate an essential role of the Rho-Rock-Myosin signaling axis for the regulation of basic cell-cell communications in both mouse and human ES cells,and would contribute to advance in medically compatible xeno-free environments for human pluripotent stem cells. View Publication

BACKGROUND: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy,although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. METHODOLOGY/FINDINGS: Treatment of lung tumor cells with doxorubicin,cisplatin,or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133,CD117,SSEA-3,TRA1-81,Oct-4,and nuclear beta-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres,maintain self-renewal capacity,and differentiate. Differentiated progenitors lost expression of CD133,gained CK 8/18 and acquired drug sensitivity. In the presence of drugs,differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice,which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF,bFGF,IL-6,IL-8,HGF,PDGF-BB,G-CSF,and SCGF-beta). CSCs also showed elevated levels of expression of human VEGFR2,FGFR2,CXCR1,2 and 4 receptors. Moreover,human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. View Publication

Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis,biological activity,and further profiling of these compounds are described. This work resulted in the discovery of 17,GDC-0941,which is a potent,selective,orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer. View Publication

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin,causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs,when cotransfected with recombinatory donor DNA fragments,can promote single base-pair modification at the start of the second intron of the beta-globin gene,the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose,sequence,cell-cycle stage,and the presence of a homologous donor DNA molecule. Enhanced recombination,with frequencies up to 0.4%,was observed with use of the lysomotropic agent chloroquine. Finally,we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells,including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable,site-specific modification of disease-related genes in human cells. 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

Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells,thus over time normalizing the hematologic status of the patient. In contrast to these observations,it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype,the FA-D1 (Brca2(Delta27/Delta27)) mice,we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data,hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia,leukemias,or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells. View Publication

Mouse embryonic fibroblasts (MEFs) deficient for pocket proteins (i.e.,pRB/p107-,pRB/p130-,or pRB/p107/p130-deficient MEFs) have lost proper G(1) control and are refractory to Ras(V12)-induced senescence. However,pocket protein-deficient MEFs expressing Ras(V12) were unable to exhibit anchorage-independent growth or to form tumors in nude mice. We show that depending on the level of pocket proteins,loss of adhesion induces G(1) and G(2) arrest,which could be alleviated by overexpression of the TBX2 oncogene. TBX2-induced transformation occurred only in the absence of pocket proteins and could be attributed to downregulation of the p53/p21(CIP1) pathway. Our results show that a balance between the pocket protein and p53 pathways determines the level of transformation of MEFs by regulating cyclin-dependent kinase activities. Since transformation of human fibroblasts also requires ablation of both pathways,our results imply that the mechanisms underlying transformation of human and mouse cells are not as different as previously claimed. View Publication

We analyzed 21 children with leukemia receiving haploidentical hematopoietic stem cell transplantation (haplo-HSCT) from killer immunoglobulin (Ig)-like receptors (KIR) ligand-mismatched donors. We showed that,in most transplantation patients,variable proportions of donor-derived alloreactive natural killer (NK) cells displaying anti-leukemia activity were generated and maintained even late after transplantation. This was assessed through analysis of donor KIR genotype,as well as through phenotypic and functional analyses of NK cells,both at the polyclonal and clonal level. Donor-derived KIR2DL1(+) NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells,including patient leukemia blasts. Differently,KIR2DL2/3(+) NK cells displayed poor alloreactivity against leukemia cells carrying human leukocyte antigen (HLA) alleles belonging to C2 group. Unexpectedly,this was due to recognition of C2 by KIR2DL2/3,as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably,however,C2/C2 leukemia blasts were killed by KIR2DL2/3(+) (or by NKG2A(+)) NK cells that coexpressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role of the KIR2DS2 activating receptor in leukemia cell lysis could not be demonstrated. Altogether,these results may have important clinical implications for the selection of optimal donors for haplo-HSCT. View Publication

The trypsin-like serine protease marapsin is a member of the large protease gene cluster at human chromosome 16p13.3,which also contains the structurally related proteases testisin,tryptase epsilon,tryptase gamma,and EOS. To gain insight into the biological functions of marapsin,we undertook a detailed gene expression analysis. It showed that marapsin expression was restricted to tissues containing stratified squamous epithelia and was absent or only weakly expressed in all other tissues,including the pancreas. Marapsin was constitutively expressed in nonkeratinizing stratified squamous epithelia of human esophagus,tonsil,cervix,larynx,and cornea. In the keratinizing stratified squamous epidermis of skin,however,its expression was induced only during epidermal hyperproliferation,such as in psoriasis and in murine wound healing. In fact,marapsin was the second most strongly up-regulated protease in psoriatic lesions,where expression was localized to the upper region of the hyperplastic epidermis. Similarly,in the hyperproliferative epithelium of regenerating murine skin wounds,marapsin localized to the suprabasal layers,where keratinocytes undergo squamous differentiation. The transient up-regulation of marapsin,which closely correlated with re-epithelialization,was virtually absent in a genetic mouse model of delayed wound closure. These results suggested a function during the process of re-epithelialization. Furthermore,in reconstituted human epidermis,a model system of epidermal differentiation,members of the IL-20 subfamily of cytokines,such as IL-22,induced marapsin expression. Consistent with a physiologic role in marapsin regulation,IL-22 was also strongly expressed in re-epithelializing skin wounds. Marapsin's restricted expression,localization,and cytokine-inducible expression suggest a role in the terminal differentiation of keratinocytes in hyperproliferating squamous epithelia. View Publication

Notch signaling regulates cell differentiation and proliferation,contributing to the maintenance of diverse tissues including the intestinal epithelia. However,its role in tissue regeneration is less understood. Here,we show that Notch signaling is activated in a greater number of intestinal epithelial cells in the inflamed mucosa of colitis. Inhibition of Notch activation in vivo using a gamma-secretase inhibitor resulted in a severe exacerbation of the colitis attributable to the loss of the regenerative response within the epithelial layer. Activation of Notch supported epithelial regeneration by suppressing goblet cell differentiation,but it also promoted cell proliferation,as shown in in vivo and in vitro studies. By utilizing tetracycline-dependent gene expression and microarray analysis,we identified a novel group of genes that are regulated downstream of Notch1 within intestinal epithelial cells,including PLA2G2A,an antimicrobial peptide secreted by Paneth cells. Finally,we show that these functions of activated Notch1 are present in the mucosa of ulcerative colitis,mediating cell proliferation,goblet cell depletion,and ectopic expression of PLA2G2A,thereby contributing to the regeneration of the damaged epithelia. This study showed the critical involvement of Notch signaling during intestinal tissue regeneration,regulating differentiation,proliferation,and antimicrobial response of the epithelial cells. Thus Notch signaling is a key intracellular molecular pathway for the proper reconstruction of the intestinal epithelia. View Publication