技术资料

Because lymphoid progenitors can give rise to natural killer (NK) cells,NK ontogeny has been considered to be exclusively lymphoid. Here,we show that rare human CD34(+) hematopoietic progenitors develop into NK cells in vitro in the presence of cytokines (interleukin-7,interleukin-15,stem cell factor,and fms-like tyrosine kinase-3 ligand). Adding hydrocortisone and stromal cells greatly increases the frequency of progenitor cells that give rise to NK cells through the recruitment of myeloid precursors,including common myeloid progenitors and granulocytic-monocytic precursors to the NK-cell lineage. WNT signaling was involved in this effect. Cells at more advanced stages of myeloid differentiation (with increasing expression of CD13 and macrophage colony-stimulating factor receptor [M-CSFR]) could also differentiate into NK cells in the presence of cytokines,stroma,and hydrocortisone. NK cells derived from myeloid precursors (CD56(-)CD117(+)M-CSFR(+)) showed more expression of killer immunoglobulin-like receptors,a fraction of killer immunoglobulin-like receptor-positive-expressing cells that lacked NKG2A,a higher cytotoxicity compared with CD56(-)CD117(+)M-CSFR(-) precursor-derived NK cells and thus resemble the CD56(dim) subset of NK cells. Collectively,these studies show that NK cells can be derived from the myeloid lineage. View Publication

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors,but the functional capacity of DC must be assessed in detail,especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant,providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol,resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors,or TLRs,do not show these functional features. Therefore,SeV-infected DC have the potential for DC-directed immunotherapy. View Publication

In elderly subjects and in patients with chronic inflammatory diseases,there is an increased subset of monocytes with a CD14(+)CD16(+) phenotype,whose origin and functional relevance has not been well characterized. In this study,we determined whether prolonged survival of human CD14(++)CD16(-) monocytes promotes the emergence of senescent cells,and we analyzed their molecular phenotypic and functional characteristics. We used an in vitro model to prolong the life span of healthy monocytes. We determined cell senescence,intracellular cytokine expression,ability to interact with endothelial cells,and APC activity. CD14(+)CD16(+) monocytes were senescent cells with shortened telomeres (215 ± 37 relative telomere length) versus CD14(++)CD16(-) cells (339 ± 44 relative telomere length; p textless 0.05) and increased expression of β-galactosidase (86.4 ± 16.4% versus 10.3 ± 7.5%,respectively; p = 0.002). CD14(+)CD16(+) monocytes exhibited features of activated cells that included expression of CD209,release of cytokines in response to low-intensity stimulus,and increased capacity to sustain lymphocyte proliferation. Finally,compared with CD14(++)CD16(-) cells,CD14(+)CD16(+) monocytes showed elevated expression of chemokine receptors and increased adhesion to endothelial cells (19.6 ± 8.1% versus 5.3 ± 4.1%; p = 0.033). In summary,our data indicated that the senescent CD14(+)CD16(+) monocytes are activated cells,with increased inflammatory activity and ability to interact with endothelial cells. Therefore,accumulation of senescent monocytes may explain,in part,the development of chronic inflammation and atherosclerosis in elderly subjects and in patients with chronic inflammatory diseases. View Publication

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species,the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest,followed by an immunodepletion step using microbeads coated with CD11b,CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion,a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1),CD90,CD105 and CD73 cell surface markers,but negative for the hematopoietic surface markers CD14,CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic,osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time. View Publication

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here,we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs.backslashnbackslashnEXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points,day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C.backslashnbackslashnKEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2,TUBB III,PAX6,TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2,PITX2,GATA5,MYL4,TNNT2,COL1A1 and COL1A2. In addition,no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated.backslashnbackslashnCONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments. View Publication

Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP),a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase,and recapitulated,although aberrantly,in the subsequent ischemia-induced neovessel formation phase of ROP. Yet,whereas the histopathological features of ROP are well characterized,many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed,matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably,injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion,migration,and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands,their receptors,inhibitors,and downstream targets. CCN1-induced Wnt signaling mediated,at least in part,adhesion and endothelial differentiation of cultured HSCs,and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy. View Publication

With regard to human induced pluripotent stem cells (hiPSCs),in which adult cells are reprogrammed into embryonic-like cells using defined factors,their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study,hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method,and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs,hESCs,hiPSC-ECs,hESC-ECs,and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the distances" among hiPSCs� View Publication

BACKGROUND: Primitive brain tumors are the leading cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs),thought to account for tumorigenesis and therapeutic resistance,have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. METHODOLOGY/PRINCIPAL FINDINGS: Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples,regardless of their histopathologies and grades of malignancy (57% of embryonal tumors,57% of low-grade gliomas and neuro-glial tumors,70% of ependymomas,91% of high-grade gliomas). Most high-grade glioma-derived oncospheres (10/12) sustained long-term self-renewal akin to neural stem cells (textgreater7 self-renewals),whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumors. Regardless of tumor entities,the young age group was associated with self-renewal properties akin to neural stem cells (P = 0.05,chi-square test). Survival analysis of the cohort showed an association between isolation of cells with long-term self-renewal abilities and a higher patient mortality rate (P = 0.013,log-rank test). Sampling of low- and high-grade glioma cultures showed that self-renewing cells forming oncospheres shared a molecular profile comprising embryonic and neural stem cell markers. Further characterization performed on subsets of high-grade gliomas and one low-grade glioma culture showed combination of this profile with mesenchymal markers,the radio-chemoresistance of the cells and the formation of aggressive tumors after intracerebral grafting. CONCLUSIONS/SIGNIFICANCE: In brain tumors affecting adult patients,TSCs have been isolated only from high-grade gliomas. In contrast,our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors. View Publication

Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are promising can- didate cell sources for regenerative medicine. However,despite the common ability of hiPSCs and hESCs to dif- ferentiate into all 3 germ layers,their functional equivalence at the single cell level remains to be demonstrated. Moreover,single cell heterogeneity amongst stem cell populations may underlie important cell fate decisions. Here,we used single cell analysis to resolve the gene expression profiles of 362 hiPSCs and hESCs for an array of 42 genes that characterize the pluripotent and differentiated states. Comparison between single hESCs and single hiPSCs revealed markedly more heterogeneity in gene expression levels in the hiPSCs,suggesting that hiPSCs occupy an alternate,less stable pluripotent state. hiPSCs also displayed slower growth kinetics and impaired directed differentiation as compared with hESCs. Our results suggest that caution should be exer- cised before assuming that hiPSCs occupy a pluripotent state equivalent to that of hESCs,particularly when producing differentiated cells for regenerative medicine aims. View Publication

Human embryonic stem (hES) cells have a potential use for the repair and regeneration of injured tissues. However,teratoma formation can be a major obstacle for hES-mediated cell therapy. Therefore,tracking the fate and function of transplanted hES cells with noninvasive imaging could be valuable for a better understanding of the biology and physiology of teratoma formation. In this study,hES cells were stably transduced with a double fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein. Following bioluminescence imaging and histology,we demonstrated that engraftment of hES cells was followed by dramatically increasing signaling and led to teratoma formation confirmed by histology. Studies of the angiogenic processes within teratomas revealed that their vasculatures were derived from both differentiated hES cells and host. Moreover,FACS analysis showed that teratoma cells derived from hES cells expressed high levels of CD56 and SSEA-4,and the subcultured SSEA-4(+) cells showed a similar cell surface marker expression pattern when compared to undifferentiated hES cells. We report here for the first time that SSEA-4(+) cells derived from teratoma exhibited multipotency,retained their differentiation ability in vivo as confirmed by their differentiation into representative three germ layers. View Publication

Patients with Fanconi anemia (FA) have a high risk of developing acute myeloid leukemia (AML). In this study,we attempted to identify cell-surface markers for leukemia-initiating cells in FA-AML patients. We found that the IL-3 receptor-α (IL-3Rα) is a promising candidate as an leukemia-initiating cell-specific antigen for FA-AML. Whereas IL-3Rα expression is undetectable on normal CD34(+)CD38(-) HSCs,it is overexpressed on CD34(+)CD38(-) cells from FA patients with AML. We examined the leukemia-initiating cell activity of IL-3Rα-positive FA-AML cells in a humanized" FA xenotransplant model in which we separated AML cells into IL-3Rα-positive and IL-3Rα-negative CD34 fractions and transplanted them into irradiated recipient mice. In all 3 FA-AML samples� View Publication

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature ageing disease,characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs). HGPS is caused by a single point mutation in the lamin A (LMNA) gene,resulting in the generation of progerin,a truncated splicing mutant of lamin A. Accumulation of progerin leads to various ageing-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin. Here we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin,and more importantly,lack the nuclear envelope and epigenetic alterations normally associated with premature ageing. Upon differentiation of HGPS-iPSCs,progerin and its ageing-associated phenotypic consequences are restored. Specifically,directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular ageing. Additionally,our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs,also known as PRKDC) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological ageing. Because progerin also accumulates during physiological ageing,our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular ageing. View Publication