技术资料

BACKGROUND: Excessive maturation of hematopoietic cells leads to a reduction of long-term proliferative capability during cord blood (CB) expansion. In this study,we report the effects of anit-CD52 (Alemtuzumab,Campath) on both short- and long-term ex vivo expansion of CB hematopoietic stem cells (HSC) by evaluating the potential role of Alemtuzumab in preserving the repopulating capability in CB HSC and nonlymphoid progenitors. METHODS: Ex vivo expansion experiments were carried out using freshly purified CB CD34(+)cells in StemSpantrade mark SFEM medium in the presence of stem cell factor,Flt3-Ligand and thrombopoietin at 50 ng/ml. Alemtuzumab (10 microg/ml) was used to deplete CD52(+) cells during the cultures. Flow cytometry was used to monitor CB HSC and their differentiation. Colony forming unit (CFU) assays and long term culture-initiating cell (LTC-IC) assays were performed on cells obtained from day 0 (before culture) and day 14 after cultures. Secondary cultures was performed using CD34(+) cells isolated at 35 days from primary cultures and further cultured in StemSpantrade mark SFEM medium for another 14 days to confirm the long term effect of alemtuzumab in liquid cultures. RESULTS: Compared to cytokines alone,addition of alemtuzumab resulted in a significant increase in total nucleated cells,absolute CD34(+) cells,myeloid and megakaryocytic progenitors,multi-lineage and myeloid CFU and LTC-IC. CONCLUSION: The results from current study suggested that the use of alemtuzumab for ex vivo expansion of CBHSC maybe advantageous. Our findings may improve current technologies for CBHSC expansion and increase the availability of CB units for transplantation. However,in vivo studies using animal models are likely needed in further studies to test the hematopoietic effects using such expanded CB products. View Publication

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life-threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein (MCP-1)/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific l-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in vitro and in vivo. We injected MRL(lpr/lpr) mice with DPLN from 14 weeks of age with vehicle,weekly 30 mg/kg CYC (full dose),monthly 30 mg/kg CYC (one-fourth full dose),pegylated control Spiegelmer,pegylated anti-Ccl2 Spiegelmer (3/week),pegylated anti-Ccl2 Spiegelmer plus CYC one-fourth full dose and mycophenolate mofetil. At week 24,DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC one-fourth full dose. The CYC one-fourth full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3(+)CD4(-)CD8(-) and CD3(+)CD4(+)CD25(+) T cells and serum interleukin-12p40 and tumor necrosis factor-alpha levels were all markedly affected by CYC full dose but not by CYC one-fourth full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow colony-forming unit-granulocyte macrophage counts and 7/4(high) monocyte counts,lymphoproliferation,and spleen T cell depletion. In summary,anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controlling DPLN and pneumonitis in MRL-Fas(lpr) mice,sparing suppressive effects of full-dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as a novel strategy to reduce CYC toxicity in the treatment of severe lupus. View Publication

Human embryonic stem (hES) cells represent a potential source for cell replacement therapy of many degenerative diseases. Most frequently,hES cell lines are derived from surplus embryos from assisted reproduction cycles,independent of their quality or morphology. Here,we show that hES cell lines can be obtained from poor-quality blastocysts with the same efficiency as that obtained from good- or intermediate-quality blastocysts. Furthermore,we show that the self-renewal,pluripotency,and differentiation ability of hES cell lines derived from either source are comparable. Finally,we present a simple and reproducible embryoid body-based protocol for the differentiation of hES cells into functional cardiomyocytes. The five new hES cell lines derived here should widen the spectrum of available resources for investigating the biology of hES cells and advancing toward efficient strategies of regenerative medicine. View Publication

Large-scale manufacture of human embryonic stem cells (hESCs) is prerequisite to their widespread use in biomedical applications. However,current hESC culture strategies are labor-intensive and employ highly variable processes,presenting challenges for scaled production and commercial development. Here we demonstrate that passaging of the hESC lines,HUES7,and NOTT1,with trypsin in feeder-free conditions,is compatible with complete automation on the CompacT SelecT,a commercially available and industrially relevant robotic platform. Pluripotency was successfully retained,as evidenced by consistent proliferation during serial passage,expression of stem cell markers (OCT4,NANOG,TRA1-81,and SSEA-4),stable karyotype,and multi-germlayer differentiation in vitro,including to pharmacologically responsive cardiomyocytes. Automation of hESC culture will expedite cell-use in clinical,scientific,and industrial applications. View Publication

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However,direct injection of hESCs,and cells differentiated from hESCs,into living organisms has thus far been hampered by significant cell death,teratoma formation,and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization,proliferation,and viability. Molecular imaging has given investigators a high-throughput,inexpensive,and sensitive means for tracking in vivo cell proliferation over days,weeks,and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment,proliferation,and teratoma-formation in living subjects. A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes,under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery,are introduced into cells using a variety of vector and non-vector methods. Once in the cell,reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions,depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive,noninvasive instrumentation (e.g.,CCD cameras) using signal-generating probes such as D-luciferin. To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging,bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase,derived from the firefly Photinus pyralis,encodes an enzyme that catalyzes D-luciferin to the optically active metabolite,oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells,allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore,because expression of the reporter gene product is required for signal generation,only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. In this video,the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described. View Publication

Although recent advances have enabled hematopoietic stem cells (HSCs) to be enriched to near purity,more information about their characteristics will improve our understanding of their development and stage-related functions. Here,using microarray technology,we identified endothelial cell-selective adhesion molecule (ESAM) as a novel marker for murine HSCs in fetal liver. Esam was expressed at high levels within a Rag1(-) c-kit(Hi) Sca1(+) HSC-enriched fraction,but sharply down-regulated with activation of the Rag1 locus,a valid marker for the most primitive lymphoid progenitors in E14.5 liver. The HSC-enriched fraction could be subdivided into 2 on the basis of ESAM levels. Among endothelial antigens on hematopoietic progenitors,ESAM expression showed intimate correlation with HSC activity. The ESAM(Hi) population was highly enriched for multipotent myeloid-erythroid progenitors and primitive progenitors with lymphopoietic activity,and exclusively reconstituted long-term lymphohematopoiesis in lethally irradiated recipients. Tie2(+) c-kit(+) lymphohematopoietic cells in the E9.5-10.5 aorta-gonad-mesonephros region also expressed high levels of ESAM. Furthermore,ESAM was detected on primitive hematopoietic progenitors in adult bone marrow. Interestingly,ESAM expression in the HSC-enriched fraction was up-regulated in aged mice. We conclude that ESAM marks HSC in murine fetal liver and will facilitate studies of hematopoiesis throughout life. View Publication

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. View Publication

Megakaryoblastic leukemia 1 (MKL1),identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia,is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate,overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34(+) cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down,suggesting that MKL1 acts through SRF. Consistent with these findings in human cells,knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood,and reduced ploidy in bone marrow megakaryocytes. In conclusion,MKL1 promotes physiologic maturation of human and murine megakaryocytes. View Publication

Focal adhesion kinase (FAK) has been implicated in the development of cancers,including those of the breast. Nevertheless,the molecular and cellular mechanisms by which FAK promotes mammary tumorigenesis in vivo are not well understood. Here,we show that targeted deletion of FAK in mouse mammary epithelium significantly suppresses mammary tumorigenesis in a well-characterized breast cancer model. Ablation of FAK leads to the depletion of a subset of bipotent cells in the tumor that express both luminal marker keratin 8/18 and basal marker keratin 5. Using mammary stem/progenitor markers,including aldehyde dehydrogenase,CD24,CD29,and CD61,we further revealed that ablation of FAK reduced the pool of cancer stem/progenitor cells in primary tumors of FAK-targeted mice and impaired their self-renewal and migration in vitro. Finally,through transplantation in NOD-SCID mice,we found that cancer stem/progenitor cells isolated from FAK-targeted mice have compromised tumorigenicity and impaired maintenance in vivo. Together,these results show a novel function of FAK in maintaining the mammary cancer stem/progenitor cell population and provide a novel mechanism by which FAK may promote breast cancer development and progression. View Publication

BACKGROUND: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood,ex vivo transduction of the gene of interest into them,and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor,time and money,while enhancing HSCs viability,transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes,in which reverse transcription of viral DNA is not completed. METHODS: In the present study,we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors,based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction,we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector,developed in our laboratory,that allows targeted transduction to specific cell receptors via antibody recognition. RESULTS: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs. CONCLUSIONS: Overall,the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification. View Publication

BACKGROUND: Multiple measures of endothelial progenitor cells (EPCs) have been described,but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies. METHODS: We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients,EPCs were also identified on the basis of cell surface marker expression (CD133,CD34,and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity. RESULTS: Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however,endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133,CD34,CD133/CD34,and ALDH activity correlated with each other,but not with VEGFR-2(+) cells. CONCLUSIONS: Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells,which give rise to endothelial CFUs,and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research. View Publication

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here,we show that MLL normally regulates expression of mir-196b,a hematopoietic microRNA located within the HoxA cluster,in a pattern similar to that of the surrounding 5' Hox genes,Hoxa9 and Hoxa10,during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage,mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b,while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore,overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival,as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. View Publication