技术资料

Human-induced pluripotent stem cells (iPSCs) generated from human adult somatic cells through reprogramming hold great promises for future regenerative medicine. However,exposure of human iPSCs to animal feeder and serum in the process of their generation and maintenance imposes risk of transmitting animal pathogens to human subjects,thus hindering the potential therapeutic applications. Here,we report the successful generation of human iPSCs in a feeder-independent culture system with defined factors. Two stable human iPSC lines were established from primary human dermal fibroblasts of two healthy volunteers. These human iPSCs expressed a panel of pluripotency markers including stage-specific embryonic antigen (SSEA)-4,tumor-rejection antigen (TRA)-1-60,TRA-1-81,and alkaline phosphatase,while maintaining normal karyotypes and the exogenous reprogramming factors being silenced. In addition,these human iPSCs can differentiate along lineages representative of the three embryonic germ layers upon formation of embryoid bodies,indicating their pluripotency. Furthermore,subcutaneous transplantation of these cells into immunodeficient mice resulted in teratoma formation in 6 to 8 weeks. Our findings are an important step toward generating patient-specific iPSCs in a more clinically compliant manner by eliminating the need of animal feeder cells and animal serum. View Publication

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC),we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34(+) cells. Treatment of PMF CD34(+) cells with chromatin-modifying agents (CMAs) but not hydroxyurea,Janus kinase 2 (JAK2) inhibitors,or low doses of interferon-α led to the generation of greater numbers of CD34(+) chemokine (C-X-C motif) receptor (CXCR)4(+) cells,which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F(+). Furthermore,sequential treatment of PMF CD34(+) cells but not normal CD34(+) cells with decitabine (5-aza-2'-deoxycytidine [5azaD]),followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA),or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F(+) PMF CD34(+) cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγ(null) mice,the percentage of JAK2V617F/JAK2(total) in human CD45(+) marrow cells was dramatically reduced. These findings suggest that both PMF HPCs,short-term and long-term SCID repopulating cells (SRCs),are JAK2V617F(+) and that JAK2V617F(+) HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs,therefore,represents a possible effective means of treating PMF at the level of the malignant SRC. View Publication

Different molecular markers have been identified for melanoma-initiating cells including CD133 and nestin. Assuming that metastasis requires a dissemination of tumor-initiating cells,presence of circulating tumor-initiating cells should be associated with worse patient outcome. In this study,20 ml blood was collected from 32 consecutive patients affected by metastatic melanoma and blood was enriched for circulating melanoma cells (CMCs) by CD45 depletion of the non-melanoma cell fraction. Multiparameter cytometry was carried out to co-stain with combinations of CD133 and nestin (NES). Six tissue samples from metastatic lesions of six different patients were stained with the same antibodies by immunohistochemistry. Percentage of NES-positive CMCs correlated with tumor burden and number of metastatic sites. Cox regression analysis revealed levels of lactate dehydrogenase (LDH; hazard ratio: 12.8 (1.35-121.5); P=0.02),number of metastatic sites (hazard ratio 3.87 (1.66-9.03); P=0.02),tumor burden (hazard ratio 5.72 (1.57-20.9); P=0.01),and percentage of NES-expressing CMCs ≥ 35% (hazard ratio 5.73 (1.66-19.7); P=0.006) to be factors related to shorter overall survival. CD133- and NES-expression profiles on CMCs were similar to matched metastatic tissue. These findings show that CMCs expressed stem cell-associated markers NES and CD133. Higher expression of NES on CMCs might represent an index of poor prognosis. View Publication

For human embryonic stem cells (ESC) to be used in cell replacement therapies,they must be grown under good manufacturing conditions in a chemically defined medium that lacks animal proteins. This study examined the ability of a newly designed medium containing the plant-derived serum replacement VegetaCell and other reagents of human origin to support undifferentiated growth and pluripotency of human ESC. This medium was tested in several culture systems,using human fibroblasts as a feeder layer or Matrigel in a feeder-free culture. Even under the most stringent feeder-free conditions without conditioned medium,human ESC exhibited an undifferentiated morphology,expressed markers of undifferentiated cells,demonstrated high alkaline phosphatase activity and multilineage differentiation and retained a normal karyotype. Compared with human ESC grown in standard culture conditions,human ESC maintained in humanized VegetaCell medium show longer cell cycles and decreased cell death. The availability of an animal protein-free medium supplemented with the low-cost VegetaCell reagent expands the repertoire of media for culturing human ESC as well as induced pluripotent stem cells for drug testing and cell replacement therapy. View Publication

BACKGROUND: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However,since in most of the studies reported in literature the engraftment state was observed in the nucleated cells,in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. DESIGN AND METHODS: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C,-c,-D,-E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation. RESULTS: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%,46%,15% and 25% at day 1364,1385,1314 and 932,respectively. Similar results were obtained for the erythroid precursors,analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast,on the same days of observation,the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%,100%,73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant,although the biological mechanisms underlying these observations need further investigation. View Publication

High maternal blood glucose levels caused by diabetes mellitus can irreversibly lead to maldevelopment of the growing fetus with specific effects on the skeleton. To date,it remains controversial at which stage embryonic development is affected. Specifically during embryonic bone development,it is unclear whether diminished bone mineral density is caused by reduced osteoblast or rather enhanced osteoclast function. Therefore,the aim of this study was to characterize the growth as well as the skeletal differentiation capability of pluripotent embryonic stem cells (ESCs),which may serve as an in vitro model for all stages of embryonic development,when cultured in diabetic levels of D-glucose (4.5 g/L) versus physiological levels (1.0 g/L). Results showed that cells cultivated in physiological glucose gave rise to a higher number of colonies with an undifferentiated character as compared to cells grown in diabetic glucose concentrations. In contrast,these cultures were characterized by slightly decreased expression of proteins associated with the stem cell state. Furthermore,differentiation of ESCs into osteoblasts and osteoclasts was favored in physiological glucose concentrations,demonstrated by an increased matrix calcification,enhanced expression of cell-type-specific mRNAs,as well as activity of the cell-type-specific enzymes,alkaline,and tartrate resistant acidic phosphatase. In fact,this pattern was noted in murine as well as in primate ESCs. Our study suggests that an interplay between both the osteoblast and the osteoclast lineage is needed for proper skeletal development to occur,which seems impaired in hyperglycemic conditions. View Publication

Thyroid carcinoma is the most common endocrine malignancy and the first cause of death among endocrine cancers. We show that the tumorigenic capacity in thyroid cancer is confined in a small subpopulation of stem-like cells with high aldehyde dehydrogenase (ALDH(high)) activity and unlimited replication potential. ALDH(high) cells can be expanded indefinitely in vitro as tumor spheres,which retain the tumorigenic potential upon delivery in immunocompromised mice. Orthotopic injection of minute numbers of thyroid cancer stem cells recapitulates the behavior of the parental tumor,including the aggressive metastatic features of undifferentiated thyroid carcinomas,which are sustained by constitutive activation of cMet and Akt in thyroid cancer stem cells. The identification of tumorigenic and metastagenic thyroid cancer cells may provide unprecedented preclinical tools for development and preclinical validation of novel targeted therapies. View Publication

The longevity of organisms is maintained by stem cells. If an organism loses the ability to maintain a balance between quiescence and differentiation in the stem/progenitor cell compartment due to aging and/or stress,this may result in death or age-associated diseases,including cancer. Ewing sarcoma is the most lethal bone tumor in young patients and arises from primitive stem cells. Here,we demonstrated that endogenous Ewing sarcoma gene (Ews) is indispensable for stem cell quiescence,and that the ablation of Ews promotes the early onset of senescence in hematopoietic stem progenitor cells. The phenotypic and functional changes in Ews-deficient stem cells were accompanied by an increase in senescence-associated β-galactosidase staining and a marked induction of p16(INK4a) compared with wild-type counterparts. With its relevance to cancer and possibly aging,EWS is likely to play a significant role in maintaining the functional capacity of stem cells and may provide further insight into the complexity of Ewing sarcoma in the context of stem cells. View Publication

Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998),human embryonic stem cells (hESCs) have been derived from blastocysts,and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs,fibroblast feeder layers are often used at some phase in the culturing protocol. The feeders - often mouse embryonic fibroblasts (mEFs) - provide a substrate that increases plating efficiency,helps maintain pluripotency,and facilitates survival and growth of the stem cells. Various protocols for culturing embryonic stem cells from both species are available with newer trends moving toward feeder-free and serum-free culture. The purpose of this chapter is to provide basic protocol information on the isolation of mouse embryonic fibroblasts and establishment of feeder layers,the culture of mESCs on both mEFs and on gelatin in serum-containing medium,and the culture of hESCs in defined media on both mEFs (hESC culture medium) and Matrigel (mTeSR). These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose. View Publication

Human neural progenitor cells provide a source for cell replacement therapy to treat neurodegenerative diseases. Therefore,there is great interest in mechanisms and tools to direct the fate of multipotent progenitor cells during their differentiation to increase the yield of a desired cell type. We tested small molecule inhibitors of glycogen synthase kinase-3 (GSK-3) for their functionality and their influence on neurogenesis using the human neural progenitor cell line ReNcell VM. Here we report the enhancement of neurogenesis of human neural progenitor cells by treatment with GSK-3 inhibitors. We tested different small molecule inhibitors of GSK-3 i.e. LiCl,sodium-valproate,kenpaullone,indirubin-3-monoxime and SB-216763 for their ability to inhibit GSK-3 in human neural progenitor cells. The highest in situ GSK-3 inhibitory effect of the drugs was found for kenpaullone and SB-216763. Accordingly,kenpaullone and SB-216763 were the only drugs tested in this study to stimulate the Wnt/β-catenin pathway that is antagonized by GSK-3. Analysis of human neural progenitor differentiation revealed an augmentation of neurogenesis by SB-216763 and kenpaullone,without changing cell cycle exit or cell survival. Small molecule inhibitors of GSK-3 enhance neurogenesis of human neural progenitor cells and may be used to direct the differentiation of neural stem and progenitor cells in therapeutic applications. View Publication

The conversion of lineage-committed cells to induced pluripotent stem cells (iPSCs) by reprogramming is accompanied by a global remodeling of the epigenome,resulting in altered patterns of gene expression. Here we characterize the transcriptional reorganization of large intergenic non-coding RNAs (lincRNAs) that occurs upon derivation of human iPSCs and identify numerous lincRNAs whose expression is linked to pluripotency. Among these,we defined ten lincRNAs whose expression was elevated in iPSCs compared with embryonic stem cells,suggesting that their activation may promote the emergence of iPSCs. Supporting this,our results indicate that these lincRNAs are direct targets of key pluripotency transcription factors. Using loss-of-function and gain-of-function approaches,we found that one such lincRNA (lincRNA-RoR) modulates reprogramming,thus providing a first demonstration for critical functions of lincRNAs in the derivation of pluripotent stem cells. View Publication

In hereditary hemochromatosis,mutations in HFE lead to iron overload through abnormally low levels of hepcidin. In addition,HFE potentially modulates cellular iron uptake by interacting with transferrin receptor,a crucial protein during erythropoiesis. However,the role of HFE in this process was never explored. We hypothesize that HFE modulates erythropoiesis by affecting dietary iron absorption and erythroid iron intake. To investigate this,we used Hfe-KO mice in conditions of altered dietary iron and erythropoiesis. We show that Hfe-KO mice can overcome phlebotomy-induced anemia more rapidly than wild-type mice (even when iron loaded). Second,we evaluated mice combining the hemochromatosis and β-thalassemia phenotypes. Our results suggest that lack of Hfe is advantageous in conditions of increased erythropoietic activity because of augmented iron mobilization driven by deficient hepcidin response. Lastly,we demonstrate that Hfe is expressed in erythroid cells and impairs iron uptake,whereas its absence exclusively from the hematopoietic compartment is sufficient to accelerate recovery from phlebotomy. In summary,we demonstrate that Hfe influences erythropoiesis by 2 distinct mechanisms: limiting hepcidin expression under conditions of simultaneous iron overload and stress erythropoiesis,and impairing transferrin-bound iron uptake by erythroid cells. Moreover,our results provide novel suggestions to improve the treatment of hemochromatosis. View Publication