产品说明

PURPOSE: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However,one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example,hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens,but in vivo models to assess potential toxicities have not been developed. In this study,a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. EXPERIMENTAL DESIGN: A proof-of-concept,temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/Sz (NOD/SCID/γchain(null)),reconstituted with human hematopoietic cells,and the impact of treatment on human hematopoiesis was evaluated. RESULTS: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow,flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor,lymphoid,and myeloid). CONCLUSIONS: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. View Publication

A population of CD133(+)Lin(-)CD45(-) very small embryonic/epiblast-like stem cells (VSELs) has been purified by multiparameter sorting from umbilical cord blood (UCB). To speed up isolation of these cells,we employed anti-CD133-conjugated paramagnetic beads followed by staining with Aldefluor to detect aldehyde dehydrogenase (ALDH) activity; we subsequently sorted CD45(-)/GlyA(-)/CD133(+)/ALDH(high) and CD45(-)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for VSELs,and CD45(+)/GlyA /CD133(+)/ALDH(high) and CD45(+)/GlyA(-)/CD133(+)/ALDH(low) cells,which are enriched for hematopoietic stem/progenitor cells (HSPCs). Although freshly isolated CD45(-) VSELs did not grow hematopoietic colonies,the same cells,when activated/expanded over OP9 stromal support,acquired hematopoietic potential and grew colonies composed of CD45(+) hematopoietic cells in methylcellulose cultures. We also observed that CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs grew colonies earlier than CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs,which suggests that the latter cells need more time to acquire hematopoietic commitment. In support of this possibility,real-time polymerase chain reaction analysis confirmed that,whereas freshly isolated CD45(-)/GlyA(-)/CD133(+)/ALDH(high) VSELs express more hematopoietic transcripts (for example,c-myb),CD45(-)/GlyA(-)/CD133(+)/ALDH(low) VSELs exhibit higher levels of pluripotent stem cell markers (for example,Oct-4). More importantly,hematopoietic cells derived from VSELs that were co-cultured over OP9 support were able to establish human lympho-hematopoietic chimerism in lethally irradiated non-obese diabetic/severe combined immunodeficiency mice 4-6 weeks after transplantation. Overall,our data suggest that UCB-VSELs correspond to the most primitive population of HSPCs in UCB. View Publication

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope,mostly the lamins. Lamins are the main constituents of the nuclear lamina,a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription,DNA replication and chromatin organization. More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes,understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging,atypical Werner syndrome and Hutchinson Gilford progeria,all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype,reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases. View Publication

Age-related macular degeneration (AMD) is one of the major causes of blindness in aging population that progresses with death of retinal pigment epithelium (RPE) and photoreceptor degeneration inducing impairment of central vision. Discovery of human induced pluripotent stem (hiPS) cells has opened new avenues for the treatment of degenerative diseases using patient-specific stem cells to generate tissues and cells for autologous cell-based therapy. Recently,RPE cells were generated from hiPS cells. However,there is no evidence that those hiPS-derived RPE possess specific RPE functions that fully distinguish them from other types of cells. Here,we show for the first time that RPE generated from hiPS cells under defined conditions exhibit ion transport,membrane potential,polarized vascular endothelial growth factor secretion,and gene expression profile similar to those of native RPE. The hiPS-RPE could therefore be a very good candidate for RPE replacement therapy in AMD. However,these cells show rapid telomere shortening,DNA chromosomal damage,and increased p21 expression that cause cell growth arrest. This rapid senescence might affect the survival of the transplanted cells in vivo and therefore,only the very early passages should be used for regeneration therapies. Future research needs to focus on the generation of safe" as well as viable hiPS-derived somatic cells." View Publication

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media,attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component,as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces,we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives,and should be applicable to other reprogramming methods. View Publication

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However,little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis,and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4,Sox2,C-myc,and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3,STRA8,and germ cell marker-VASA,and haploid marker-FE-J1,PRM1,indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However,most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice,and showed that mGSCs can colonize,self-renew,and differentiate into germ cells. Thus,we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro,providing a promising platform for generation of transgenic goat for production of specific humanized proteins. View Publication

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes,there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs,and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore,the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control,we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis,leading to a loss of LSCs. Together,these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia. View Publication

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection,whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work,we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death,beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress,and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of,but not prevent,BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2,IAP2,MCL-1,and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased,whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus,we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS. View Publication

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article,we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule,E-cadherin,to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin,but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase,suggesting that E-cadherin promotes rapid neuronal differentiation. Further,these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin,neurofilament,and neural cell adhesion molecule. Moreover,blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation,we are able to utilize a specific cell-cell adhesion molecule,E-cadherin,to influence the nature and degree of neural specialization. View Publication

Overexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors,including the clonal PIGA(-) cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs),and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3' untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3'UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells,hypercellular bone marrow (BM),splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus. View Publication

Nature Methods 8,293 (2011). doi:10.1038/nmeth0411-293 View Publication