技术资料

The mechanisms underlying deregulation of HOX gene expression in AML are poorly understood. The ParaHox gene CDX2 was shown to act as positive upstream regulator of several HOX genes. In this study,constitutive expression of Cdx2 caused perturbation of leukemogenic Hox genes such as Hoxa10 and Hoxb8 in murine hematopoietic progenitors. Deletion of the N-terminal domain of Cdx2 abrogated its ability to perturb Hox gene expression and to cause acute myeloid leukemia (AML) in mice. In contrast inactivation of the putative Pbx interacting site of Cdx2 did not change the leukemogenic potential of the gene. In an analysis of 115 patients with AML,expression levels of CDX2 were closely correlated with deregulated HOX gene expression. Patients with normal karyotype showed a 14-fold higher expression of CDX2 and deregulated HOX gene expression compared with patients with chromosomal translocations such as t(8:21) or t(15;17). All patients with AML with normal karyotype tested were negative for CDX1 and CDX4 expression. These data link the leukemogenic potential of Cdx2 to its ability to dysregulate Hox genes. They furthermore correlate the level of CDX2 expression with HOX gene expression in human AML and support a potential role of CDX2 in the development of human AML with aberrant Hox gene expression. View Publication

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. 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

OBJECTIVE Gastric cancer (GC) is one of the most common human cancers. A useful method of gastric cancer stem cell (CSC) characterization is spheroid colony formation. Previously,we reported that KIF11 expression is textgreater2-fold in spheroid-body-forming GC cells compared with parental cells. Here,we analyzed the expression and distribution of KIF11 in human GC by immunohistochemistry. METHODS Expression of KIF11 in 165 GC cases was determined using immunohistochemistry. For mucin phenotypic expression analysis of GC,immunostaining of MUC5AC,MUC6,MUC2 and CD10 was evaluated. RNA interference was used to inhibit KIF11 expression in GC cell lines. RESULTS In total,119 of 165 GC cases (72%) were positive for KIF11. Expression of KIF11 was not associated with any clinicopathologic characteristics; however,it was observed frequently in GC exhibiting an intestinal phenotype. Both the number and size of spheres formed by MKN-74 cells were significantly reduced following transfection of KIF11-targeting siRNA compared with negative-control siRNA. Furthermore,levels of phosphorylated Erk1/2 were lower in KIF11 siRNA-transfected cells than with negative-control siRNA-transfected cells. CONCLUSION These results indicate that KIF11 is involved in intestinal mucin phenotype GC. View Publication

Human embryonic stem cells (hESCs) are pluripotent and capable of generating new $\$-cells,but current in vitro differentiation protocols generally fail to produce mature,glucose-responsive,unihormonal $\$-cells. Instead,these methods tend to produce immature polyhormonal endocrine cells which mature in vivo into glucagon-positive $\$-cells. PAX4 is an established transcription factor in $\$-cell development and function,and is capable of converting glucagon-positive cells to insulin-positive cells in mice. Work in human and mouse ESCs has shown that constitutive PAX4 expression promotes the development of insulin-positive cells,but whether acute PAX4 expression is sufficient to guide specific endocrine cell fates has not been addressed in hESCs. In this study,we applied recombinant adenovirus to ectopically express human PAX4 in hESC-derived pancreatic progenitors,with the aim of influencing the endocrine developmental cascade away from polyhormonal cells toward unihormonal insulin-positive cells. Gene delivery to pancreatic progenitors was efficient and dose-dependent. By the end of in vitro differentiation,PAX4 reduced ARX expression,but only the high dose tested significantly reduced glucagon release. Single cell analysis revealed that while PAX4 did not alter the proportion of endocrine cells,it did reduce the number of glucagon-positive cells and increased the number of unihormonal insulin-positive cells. These data suggest that acute PAX4 overexpression can reduce expression of ARX and glucagon resulting in improved numbers of unihormonal insulin-positive cells. View Publication

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD),a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production,reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs,with subsequent selection and full characterization to ensure no off-target changes,holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs." View Publication

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet,cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation,viability and differentiation potential. Here,a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation,mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover,the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics. View Publication

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition,we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated textgreater200-fold in irradiated tissues. Loss of p21 function resulted in a textgreater4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage,including 6-fold less scarring,40% increased growth potential,and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (Ptextless0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation,4-fold greater proliferation rate,and nearly 7-fold lower senescence as compared to WT MSCs (Ptextless0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition,p21 is a critical regulator of MSC proliferation,differentiation,and senescence both at baseline and in response to radiation. View Publication

The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. Here,we test whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML),an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. Our approach combined mosaic mouse models,Cre-lox technology,and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. We show that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML,while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells,such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently,myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells,resembling cancer stem cells capable of maintaining AML in vivo. Our results establish an efficient new strategy for interrogating oncogene cooperation,and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity. View Publication

Multiple studies show that tumor suppressor p53 is a barrier to dedifferentiation; whether this is strictly due to repression of proliferation remains a subject of debate. Here,we show that p53 plays an active role in promoting differentiation of human embryonic stem cells (hESCs) and opposing self-renewal by regulation of specific target genes and microRNAs. In contrast to mouse embryonic stem cells,p53 in hESCs is maintained at low levels in the nucleus,albeit in a deacetylated,inactive state. In response to retinoic acid,CBP/p300 acetylates p53 at lysine 373,which leads to dissociation from E3-ubiquitin ligases HDM2 and TRIM24. Stabilized p53 binds CDKN1A to establish a G(1) phase of cell cycle without activation of cell death pathways. In parallel,p53 activates expression of miR-34a and miR-145,which in turn repress stem cell factors OCT4,KLF4,LIN28A,and SOX2 and prevent backsliding to pluripotency. Induction of p53 levels is a key step: RNA-interference-mediated knockdown of p53 delays differentiation,whereas depletion of negative regulators of p53 or ectopic expression of p53 yields spontaneous differentiation of hESCs,independently of retinoic acid. Ectopic expression of p53R175H,a mutated form of p53 that does not bind DNA or regulate transcription,failed to induce differentiation. These studies underscore the importance of a p53-regulated network in determining the human stem cell state. View Publication

Human pluripotent stem cells (hPSCs) are sensitive to DNA damage and undergo rapid apoptosis compared to their differentiated progeny cells. Here,we explore the underlying mechanisms for the increased apoptotic sensitivity of hPSCs that helps to determine pluripotent stem cell fate. Apoptosis was induced by exposure to actinomycin D,etoposide,or tunicamycin,with each agent triggering a distinct apoptotic pathway. We show that hPSCs are more sensitive to all three types of apoptosis induction than are lineage-non-specific,retinoic-acid-differentiated hPSCs. Also,Bax activation and pro-apoptotic mitochondrial intermembrane space protein release,which are required to initiate the mitochondria-mediated apoptosis pathway,are more rapid in hPSCs than in retinoic-acid-differentiated hPSCs. Surprisingly,Bak and not Bax is essential for actinomycin-D-induced apoptosis in human embryonic stem cells. Finally,P53 is degraded rapidly in an ubiquitin-proteasome-dependent pathway in hPSCs at steady state but quickly accumulates and induces apoptosis when Mdm2 function is impaired. Rapid degradation of P53 ensures the survival of healthy hPSCs but avails these cells for immediate apoptosis upon cellular damage by P53 stabilization. Altogether,we provide an underlying,interconnected molecular mechanism that primes hPSCs for quick clearance by apoptosis to eliminate hPSCs with unrepaired genome alterations and preserves organismal genomic integrity during the early critical stages of human embryonic development. View Publication

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC,from both human and murine origin,has been reported in many studies. MSC transformation depends on the culture conditions,the origin of the cells and the time on culture; however,the precise biological characteristics involved in this process have not been fully defined yet. In this study,we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate,a shorter doubling time and also morphologic and phenotypic changes,as compared to MSC derived from wild-type animals. Furthermore,the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition,not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover,genomic instability,changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition,the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC. View Publication