技术资料

BACKGROUND: Induced pluripotent stem (iPS) cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming,which uses a defined set of transcription factors,iPS cells represent important sources of patient-specific cells for clinical applications. However,before these cells can be used in therapeutic designs,it is essential to understand their genetic stability. METHODOLOGY/PRINCIPAL FINDINGS: Here,we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation,iPS cells activate checkpoint signaling,evidenced by phosphorylation of ATM,NBS1,CHEK2,and TP53,localization of ATM to the double strand breaks (DSB),and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2) phase of the cell cycle,displaying a lack of the G(1)/S cell cycle arrest similar to human embryonic stem (ES) cells. Furthermore,both cell types remove DSB within six hours of γ-irradiation,form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally,we report elevated expression of genes involved in DNA damage signaling,checkpoint function,and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts. CONCLUSIONS/SIGNIFICANCE: High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage,dramatic changes in cell cycle structure,including a high percentage of cells in the S phase,increased radiosensitivity and loss of DNA damage-induced G(1)/S cell cycle arrest,were observed in stem cells generated by induced pluripotency. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly,bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros,whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3,as an extrinsic factor,thus supports the stemness of HSCs in the bone marrow niche. View Publication

The recent discovery of induced pluripotent stem cell (iPSC) technology provides an invaluable tool for creating in vitro representations of human genetic conditions. This is particularly relevant for those diseases that lack adequate animal models or where the species comparison is difficult,e.g. imprinting diseases such as the neurogenetic disorder Prader-Willi syndrome (PWS). However,recent reports have unveiled transcriptional and functional differences between iPSCs and embryonic stem cells that in cases are attributable to imprinting errors. This has suggested that human iPSCs may not be useful to model genetic imprinting diseases. Here,we describe the generation of iPSCs from a patient with PWS bearing a partial translocation of the paternally expressed chromosome 15q11-q13 region to chromosome 4. The resulting iPSCs match all standard criteria of bona fide reprogramming and could be readily differentiated into tissues derived from the three germ layers,including neurons. Moreover,these iPSCs retain a high level of DNA methylation in the imprinting center of the maternal allele and show concomitant reduced expression of the disease-associated small nucleolar RNA HBII-85/SNORD116. These results indicate that iPSCs may be a useful tool to study PWS and perhaps other genetic imprinting diseases as well. View Publication

Induced pluripotent stem (iPS) cells derived from terminally differentiated human fibroblasts are reprogrammed to possess stem cell like properties. However,the extent to which iPS cells exhibit unique properties of the human embryonic stem (hES) cell cycle remains to be established. hES cells are characterized by an abbreviated G1 phase (∼ 2.5 h) and accelerated organization of subnuclear domains that mediate the assembly of regulatory machinery for histone gene expression [i.e.,histone locus bodies (HLBs)]. We therefore examined cell cycle parameters of iPS cells in comparison to hES cells. Analysis of DNA synthesis [5-bromo-2'-deoxy-uridine (BrdU) incorporation],cell cycle distribution (FACS analysis and Ki67 staining) and subnuclear organization of HLBs [immunofluorescence microscopy and fluorescence in situ hybridization (FISH)] revealed that human iPS cells have a short G1 phase (∼ 2.5 h) and an abbreviated cell cycle (16-18 h). Furthermore,HLBs are formed and reorganized rapidly after mitosis (within 1.5-2 h). Thus,reprogrammed iPS cells have cell cycle kinetics and dynamic subnuclear organization of regulatory machinery that are principal properties of pluripotent hES cells. Our findings support the concept that the abbreviated cell cycle of hES and iPS cells is functionally linked to pluripotency. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date,it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development,it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore,the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs),which may serve as an in vitro model for all stages of embryonic development,when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast,these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore,differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations,demonstrated by an increased matrix calcification,enhanced expression of cell-type-specific mRNAs,as well as activity of the cell-type-specific enzymes,alkaline,and tartrate resistant acidic phosphatase. In fact,this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur,which seems impaired in hyperglycemic conditions. View Publication

BACKGROUND: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However,since in most of the studies reported in literature the engraftment state was observed in the nucleated cells,in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. DESIGN AND METHODS: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C,-c,-D,-E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation. RESULTS: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%,46%,15% and 25% at day 1364,1385,1314 and 932,respectively. Similar results were obtained for the erythroid precursors,analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast,on the same days of observation,the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%,100%,73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant,although the biological mechanisms underlying these observations need further investigation. View Publication

Although aldehyde dehydrogenase (ALDH) activity has become a surrogate of hematopoietic stem and progenitor cells (HSPCs),its function during hematopoiesis was unclear. Here,we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. Zebrafish embryos were treated with diethylaminobenzaldehyde (DEAB,1 μmol/l) between 0- and 48 hour-post-fertilization (hpf). MOs targeting aldhs were injected between 1 and 4-cell stage. The effects on hematopoiesis were evaluated at different stages. DEAB treatment between 0 and 18 hpf increased gene expression associated with HSPC (scl,lmo2),erythropoiesis (gata1,α- and β-eHb) and myelopoiesis (spi1) as well as gfp(+) cells in dissociated Tg(gata1:gfp) embryos. The effects were ameliorated by all-trans retinoic acid (1 nmol/l). Definitive hematopoiesis and the erythromyeloid precursors were unaffected. In all,14 out of 15 zebrafish aldhs were detectable by reverse transcription PCR in 18 hpf embryos,of which only aldh1a2 and aldh16a1 were expressed in sites pertinent to hematopoiesis. Molecular targeting by MOs was demonstrated for 15 aldhs,but none of them,even in combined aldh1a2 and aldh1a3 knockdown,recapitulated the hematopoietic expansion in DEAB-treated embryos. In conclusion,DEAB expands HSPC population during primitive hematopoiesis through inhibition of aldh and retinoic acid synthesis. The specific aldh isoform(s) remains to be determined. View Publication

Ectopic expression of CAAT/enhancer binding protein α (C/EBPα) in p210BCR/ABL-expressing cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects,C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5'-flanking region and enhances the promoter activity of Gfi-1. Moreover,in K562 cells,RNA interference-mediated downregulation of Gfi-1 expression partially rescued the proliferation-inhibitory but not the differentiation-inducing effect of C/EBPα. Ectopic expression of wild-type Gfi-1,but not of a transcriptional repressor mutant (Gfi-1P2A),inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast,Gfi-1 short hairpin RNA-tranduced CD34(+) chronic myeloid leukemia cells were markedly more clonogenic than the scramble-transduced counterpart. Together,these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival-inhibitory effects in BCR/ABL-expressing cells. View Publication

We have previously shown that the plant-derived compound parthenolide (PTL) can impair the survival and leukemogenic activity of primary human acute myeloid leukemia (AML) stem cells. However,despite the activity of this agent,PTL also induces cellular protective responses that likely function to reduce its overall cytotoxicity. Thus,we sought to identify pharmacologic agents that enhance the antileukemic potential of PTL. Toward this goal,we used the gene expression signature of PTL to identify compounds that inhibit cytoprotective responses by performing chemical genomic screening of the Connectivity Map database. This screen identified compounds acting along the phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways. Compared with single agent treatment,exposure of AML cells to the combination of PTL and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitors significantly decreased viability of AML cells and reduced tumor burden in vitro and in murine xenotransplantation models. Taken together,our data show that rational drug combinations can be identified using chemical genomic screening strategies and that inhibition of cytoprotective functions can enhance the eradication of primary human AML cells. View Publication

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover,safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple,nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe,efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research,disease modeling,and regenerative medicine. ?? 2010 Elsevier Inc. View Publication