技术资料

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

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

PURPOSE: The purpose of this study was to determine whether histone deacetylase (HDAC) inhibitors (HDACI) such as vorinostat or entinostat (SNDX-275) could increase the lethality of the dual Bcr/Abl-Aurora kinase inhibitor KW-2449 in various Bcr/Abl(+) human leukemia cells,including those resistant to imatinib mesylate (IM). EXPERIMENTAL DESIGN: Bcr/Abl(+) chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cells,including those resistant to IM (T315I,E255K),were exposed to KW-2449 in the presence or absence of vorinostat or SNDX-275,after which apoptosis and effects on signaling pathways were examined. In vivo studies combining HDACIs and KW2449 were carried out by using a systemic IM-resistant ALL xenograft model. RESULTS: Coadministration of HDACIs synergistically increased KW-2449 lethality in vitro in multiple CML and Ph(+) ALL cell types including human IM resistant cells (e.g.,BV-173/E255K and Adult/T315I). Combined treatment resulted in inactivation of Bcr/Abl and downstream targets (e.g.,STAT5 and CRKL),as well as increased reactive oxygen species (ROS) generation and DNA damage (γH2A.X). The latter events and cell death were significantly attenuated by free radical scavengers (TBAP). Increased lethality was also observed in primary CD34(+) cells from patients with CML,but not in normal CD34(+) cells. Finally,minimally active vorinostat or SNDX275 doses markedly increased KW2449 antitumor effects and significantly prolonged the survival of murine xenografts bearing IM-resistant ALL cells (BV173/E255K). CONCLUSIONS: HDACIs increase KW-2449 lethality in Bcr/Abl(+) cells in association with inhibition of Bcr/Abl,generation of ROS,and induction of DNA damage. This strategy preferentially targets primary Bcr/Abl(+) hematopoietic cells and exhibits enhanced in vivo activity. Combining KW-2449 with HDACIs warrants attention in IM-resistant Bcr/Abl(+) leukemias. 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

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

Identification of cancer stem cells is crucial for advancing cancer biology and therapy. Several markers including CD24,CD44,CD117,CD133,the G subfamily of ATP-binding cassette transporters (ABCG),epithelial specific antigen (ESA) and aldehyde dehydrogenase (ALDH) are used to identify and investigate human epithelial cancer stem cells in the literature. We have now systemically analyzed and compared the expression of these markers in fresh ovarian epithelial carcinomas. Although the expression levels of these markers were unexpectedly variable and partially overlapping in fresh ovarian cancer cells from different donors,we reliably detected important levels of CD133 and ALDH in the majority of fresh ovarian cancer. Furthermore,most of these stem cell markers including CD133 and ALDH were gradually lost following in vitro passage of primary tumor cells. However,the expression of ALDH and CD133,but not CD24,CD44 and CD117,could be partially rescued by the in vitro serum-free and sphere cultures and by the in vivo passage in the immune-deficient xenografts. ALDH+ and CD133+ cells formed three-dimensional spheres more efficiently than their negative counterparts. These sphere-forming cells expressed high levels of stem cell core gene transcripts and could be expanded and form additional spheres in long-term culture. ALDH+,CD133+ and ALDH+ CD133+ cells from fresh tumors developed larger tumors more rapidly than their negative counterparts. This property was preserved in the xenografted tumors. Altogether,the data suggest that ALDH+ and CD133+ cells are enriched with ovarian cancer-initiating (stem) cells and that ALDH and CD133 may be widely used as reliable markers to investigate ovarian cancer stem cell biology. 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

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

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

To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation,colony formation,and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (pulmospheres") were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability� View Publication

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

The embryonic stem cell-specific cell cycle-regulating (ESCC) family of microRNAs (miRNAs) enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Here we show that the human ESCC miRNA orthologs hsa-miR-302b and hsa-miR-372 promote human somatic cell reprogramming. Furthermore,these miRNAs repress multiple target genes,with downregulation of individual targets only partially recapitulating the total miRNA effects. These targets regulate various cellular processes,including cell cycle,epithelial-mesenchymal transition (EMT),epigenetic regulation and vesicular transport. ESCC miRNAs have a known role in regulating the unique embryonic stem cell cycle. We show that they also increase the kinetics of mesenchymal-epithelial transition during reprogramming and block TGFβ-induced EMT of human epithelial cells. These results demonstrate that the ESCC miRNAs promote dedifferentiation by acting on multiple downstream pathways. We propose that individual miRNAs generally act through numerous pathways that synergize to regulate and enforce cell fate decisions. View Publication