技术资料

The reprogramming of somatic cells into induced pluripotent stem (iPS) cells upon overexpression of the transcription factors Oct4,Sox2,Klf4 and cMyc is inefficient. It has been assumed that the somatic differentiation state provides a barrier for efficient reprogramming; however,direct evidence for this notion is lacking. Here,we tested the potential of mouse hematopoietic cells at different stages of differentiation to be reprogrammed into iPS cells. We show that hematopoietic stem and progenitor cells give rise to iPS cells up to 300 times more efficiently than terminally differentiated B and T cells do,yielding reprogramming efficiencies of up to 28%. Our data provide evidence that the differentiation stage of the starting cell has a critical influence on the efficiency of reprogramming into iPS cells. Moreover,we identify hematopoietic progenitors as an attractive cell type for applications of iPS cell technology in research and therapy. View Publication

Remarkably,apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice,but not other strains of mice,to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins,Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1,but not Bcl-2,Bcl-X(L),Bax,A1,or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand,Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis. View Publication

Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The Ras/Raf-1/MEK/ERK pathway is constitutively activated in Bcr-Abl-transformed cells,and Ras activity enhances the oncogenic ability of Bcr-Abl. However,the mechanism by which Bcr-Abl activates the Ras pathway is not completely understood. Raf kinase inhibitor protein (RKIP) inhibits activation of MEK by Raf-1 and its downstream signal transduction,resulting in blocking the MAP kinase pathway. In the present study,we found that RKIP was depleted in CML cells. We investigated the interaction between RKIP and Bcr-Abl in CML cell lines and Bcr-Abl(+) progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of RKIP and reduced the pERK1/2 status,resulting in inhibited proliferation of CML cells. Moreover,RKIP up-regulated cell cycle regulator FoxM1 expression,resulting in G(1) arrest via p27(Kip1) and p21(Cip1) accumulation. In colony-forming unit granulocyte,erythroid,macrophage,megakaryocyte,colony-forming unit-granulocyte macrophage,and burst-forming unit erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced RKIP and reduced FoxM1 expressions,and inhibited colony formation of Bcr-Abl(+) progenitor cells,whereas depletion of RKIP weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl(+) progenitor cells. Thus,Bcr-Abl represses the expression of RKIP,continuously activates pERK1/2,and suppresses FoxM1 expression,resulting in proliferation of CML cells. View Publication

The transcription factor T-bet (Tbx21) is critical for Th1 polarization of CD4(+) T cells. Genetic deletion of Tbx21 can cause either exacerbation or attenuation of different autoimmune diseases in animal models. In the nonobese diabetic (NOD) mouse,genetic deletion of the Ifng or the Il12b (IL-12p40) genes,which are both critical Th1 cytokines,does not reduce the incidence of autoimmune diabetes. These results suggest that autoimmune diabetes in the NOD may not be a Th1-driven disease. However,we report that Tbx21 deficiency in the NOD mouse completely blocks insulitis and diabetes due to defects both in the initiation of the anti-islet immune response and in the function of CD4(+) effector T cells. We find defective priming of naive islet-reactive T cells by the innate immune system in Tbx21(-/-) animals. By contrast to naive cells,activated islet-reactive BDC2.5 TCR-transgenic T cells do not require Tbx21 in recipient animals for efficient adoptive transfer of diabetes. However,when these BDC2.5 TCR-transgenic effector cells lack Tbx21,they are less effective at entering the pancreas and promoting diabetes than Tbx21(+/+) cells. Tbx21(-/-) regulatory T cells function normally in vitro and diabetes can be restored in Tbx21(-/-) mice by reducing regulatory T cell numbers. Thus,the absence of diabetes in the NOD.Tbx21(-/-) is due to intrinsic defects in both T cells and cells of the innate immune system paired with the relative preservation of regulatory T cell function. View Publication

Aldehyde dehydrogenase 1A1 (ALDH) activity is one hallmark of human bone marrow (BM),umbilical cord blood (UCB),and peripheral blood (PB) primitive progenitors presenting high reconstitution capacities in vivo. In this study,we have identified ALDH(+) cells within human skeletal muscles,and have analyzed their phenotypical and functional characteristics. Immunohistofluorescence analysis of human muscle tissue sections revealed rare endomysial cells. Flow cytometry analysis using the fluorescent substrate of ALDH,Aldefluor,identified brightly stained (ALDH(br)) cells with low side scatter (SSC(lo)),in enzymatically dissociated muscle biopsies,thereafter abbreviated as SMALD(+) (for skeletal muscle ALDH(+)) cells. Phenotypical analysis discriminated two sub-populations according to CD34 expression: SMALD(+)/CD34(-) and SMALD(+)/CD34(+) cells. These sub-populations did not initially express endothelial (CD31),hematopoietic (CD45),and myogenic (CD56) markers. Upon sorting,however,whereas SMALD(+)/CD34(+) cells developed in vitro as a heterogeneous population of CD56(-) cells able to differentiate in adipoblasts,the SMALD(+)/CD34(-) fraction developed in vitro as a highly enriched population of CD56(+) myoblasts able to form myotubes. Moreover,only the SMALD(+)/CD34(-) population maintained a strong myogenic potential in vivo upon intramuscular transplantation. Our results suggest that ALDH activity is a novel marker for a population of new human skeletal muscle progenitors presenting a potential for cell biology and cell therapy. View Publication

OBJECTIVE: Studies in pulmonary arteries (PA) of patients with chronic obstructive pulmonary disease (COPD) suggest that bone marrow-derived endothelial progenitor cells (CD133(+)) may infiltrate the intima and differentiate into smooth muscle cells (SMC). This study aimed to evaluate the plasticity of CD133(+) cells to differentiate into SMC and endothelial cells (EC) in both cell culture and human isolated PA. METHODS: Plasticity of granulocyte-colony stimulator factor (G-CSF)-mobilized peripheral blood CD133(+) cells was assessed in co-cultures with primary lines of human PA endothelial cells (PAEC) or SMC (PASMC) and in isolated human PA. We also evaluated if the phenotype of differentiated progenitor cells was acquired by fusion or differentiation. RESULTS: The in vitro studies demonstrated CD133(+) cells may acquire the morphology and phenotype of the cells they were co-cultured with. CD133(+) cells co-incubated with human isolated PA were able to migrate into the intima and differentiate into SMC. Progenitor cell differentiation was produced without fusion with mature cells. CONCLUSIONS: We provide evidence of plasticity of CD133(+) cells to differentiate into both endothelial cells and SMC,reinforcing the idea of their potential role in the remodeling process of PA in COPD. This process was conducted by transdifferentiation and not by cell fusion. View Publication

Recent studies support the notion that there is an intricate relationship between hematopoiesis and bone homeostasis in normal steady states. Using mice undergoing chronic inflammatory arthritis,we investigated the relationship between hematopoiesis and bone homeostasis in pathologic conditions. We demonstrate that mice undergoing chronic inflammatory arthritis displayed osteoporosis resulting from a severe defect in osteoblast function. Despite the defective osteoblast function,however,the hematopoietic stem cells from these mice exhibited normal properties in either long-term repopulation or cell cycling. Therefore,the bone-forming capacity of osteoblasts is distinct from their ability to maintain hematopoietic stem cells in chronic inflammatory conditions. View Publication

The cell-surface straight and branched repeats of N-acetyllactosamine (LacNAc) units,called poly-LacNAc chains,characterize the histo-blood group i and I antigens,respectively. The transition of straight to branched poly-LacNAc chain (i to I) is determined by the I locus,which expresses 3 IGnT transcripts,IGnTA,IGnTB,and IGnTC. Our previous investigation demonstrated that the i-to-I transition in erythroid differentiation is regulated by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha). In the present investigation,the K-562 cell line was used as a model to show that the i-to-I transition is determined by the phosphorylation status of the C/EBPalpha Ser-21 residue,with dephosphorylated C/EBPalpha Ser-21 stimulating the transcription of the IGnTC gene,consequently resulting in I branching. Results from studies using adult erythropoietic and granulopoietic progenitor cells agreed with those derived using the K-562 cell model,with lentiviral expression of C/EBPalpha in CD34(+) hematopoietic cells demonstrating that the dephosphorylated form of C/EBPalpha Ser-21 induced the expression of I antigen,granulocytic CD15,and also erythroid CD71 antigens. Taken together,these results demonstrate that the regulation of poly-LacNAc branching (I antigen) formation in erythropoiesis and granulopoiesis share a common mechanism,with dephosphorylation of the Ser-21 residue on C/EBPalpha playing the critical role. View Publication

Multiple myeloma is characterized by increased osteoclast activity that results in bone destruction and lytic lesions. With the prolonged overall patient survival achieved by new treatment modalities,additional drugs are required to inhibit bone destruction. We focused on a novel and more potent structural analog of the nonsteroidal anti-inflammatory drug etodolac,known as SDX-308,and its effects on osteoclastogenesis and multiple myeloma cells. SDX-101 is another structural analog of etodolac that is already used in clinical trials for the treatment of B-cell chronic lymphocytic leukemia (B-CLL). Compared with SDX-101,a 10-fold lower concentration of SDX-308 induced potent (60%-80%) inhibition of osteoclast formation,and a 10- to 100-fold lower concentration inhibited multiple myeloma cell proliferation. Bone resorption was completely inhibited by SDX-308,as determined in dentin-based bone resorption assays. SDX-308 decreased constitutive and RANKL-stimulated NF-kappaB activation and osteoclast formation in an osteoclast cellular model,RAW 264.7. SDX-308 effectively suppressed TNF-alpha-induced IKK-gamma and IkappaB-alpha phosphorylation and degradation and subsequent NF-kappaB activation in human multiple myeloma cells. These results indicate that SDX-308 effectively inhibits multiple myeloma cell proliferation and osteoclast activity,potentially by controlling NF-kappaB activation signaling. We propose that SDX-308 is a promising therapeutic candidate to inhibit multiple myeloma growth and osteoclast activity and that it should receive attention for further study. View Publication

Growth factor independence-1B (Gfi-1B) is a transcriptional repressor essential for erythropoiesis and megakaryopoiesis. Targeted gene disruption of GFI1B in mice leads to embryonic lethality resulting from failure to produce definitive erythrocytes,hindering the study of Gfi-1B function in adult hematopoiesis. We here show that,in humans,Gfi-1B controls the development of erythrocytes and megakaryocytes by regulating the proliferation and differentiation of bipotent erythro-megakaryocytic progenitors. We further identify in this cell population the type III transforming growth factor-beta receptor gene,TGFBR3,as a direct target of Gfi-1B. Knockdown of Gfi-1B results in altered transforming growth factor-beta (TGF-beta) signaling as shown by the increase in Smad2 phosphorylation and its inability to associate to the transcription intermediary factor 1-gamma (TIF1-gamma). Because the Smad2/TIF1-gamma complex is known to specifically regulate erythroid differentiation,we propose that,by repressing TGF-beta type III receptor (TbetaRIotaII) expression,Gfi-1B favors the Smad2/TIF1-gamma interaction downstream of TGF-beta signaling,allowing immature progenitors to differentiate toward the erythroid lineage. View Publication

The retinoid X receptor (RXR) contributes to the regulation of diverse biological pathways via its role as a heterodimeric partner of several nuclear receptors. However,RXR has no established role in the regulation of hematopoietic stem cell (HSC) fate. In this study,we sought to determine whether direct modulation of RXR signaling could impact human HSC self-renewal or differentiation. Treatment of human CD34(+)CD38(-)lin(-) cells with LG1506,a selective RXR modulator,inhibited the differentiation of HSCs in culture and maintained long-term repopulating HSCs in culture that were otherwise lost in response to cytokine treatment. Further studies revealed that LG1506 had a distinct mechanism of action in that it facilitated the recruitment of corepressors to the retinoic acid receptor (RAR)/RXR complex at target gene promoters,suggesting that this molecule was functioning as an inverse agonist in the context of this heterodimer. Interestingly,using combinatorial peptide phage display,we identified unique surfaces presented on RXR when occupied by LG1506 and demonstrated that other modulators that exhibited these properties functioned similarly at both a mechanistic and biological level. These data indicate that the RAR/RXR heterodimer is a critical regulator of human HSC differentiation,and pharmacological modulation of RXR signaling prevents the loss of human HSCs that otherwise occurs in short-term culture. View Publication

In ischemic congestive heart failure (CHF),anemia is associated with poor prognosis. Whether anemia develops in nonischemic CHF is uncertain. The hematopoietic inhibitors TNF-alpha and nitric oxide (NO) are activated in ischemic CHF. We examined whether mice with ischemic or nonischemic CHF develop anemia and whether TNF-alpha and NO are involved. We studied mice (n = 7-9 per group) with CHF either due to myocardial infarction (MI) or to overexpression of the Ca(2+)-binding protein calsequestrin (CSQ) or to induced cardiac disruption of the sarcoplasmic reticulum Ca(2+)-ATPase 2 gene (SERCA2 KO). Hematopoiesis was analyzed by colony formation of CD34(+) bone marrow cells. Hemoglobin concentration was 14.0 +/- 0.4 g/dl (mean +/- SD) in controls,while it was decreased to 10.1 +/- 0.4,9.7 +/- 0.4,and 9.6 +/- 0.3 g/dl in MI,CSQ,and SERCA2 KO,respectively (P textless 0.05). Colony numbers per 100,000 CD34(+) cells in the three CHF groups were reduced to 33 +/- 3 (MI),34 +/- 3 (CSQ),and 39 +/- 3 (SERCA2 KO) compared with 68 +/- 4 in controls (P textless 0.05). Plasma TNF-alpha nearly doubled in MI,and addition of anti-TNF-alpha antibody normalized colony formation. Inhibition of colony formation was completely abolished with blockade of endothelial NO synthase in CSQ and SERCA2 KO,but not in MI. In conclusion,the mechanism of anemia in CHF depends on the etiology of cardiac disease; whereas TNF-alpha impairs hematopoiesis in CHF following MI,NO inhibits blood cell formation in nonischemic murine CHF. View Publication