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Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However,whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model,we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-),CD34+/CD45+,and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+,Sca1(-)/Gr1+,VEGFR1+,and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial,pericyte,or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors,colocalized with the tumor vascular network,and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast,human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis. View Publication

Spinocerebellar ataxia type 2 (SCA2) is caused by triple nucleotidebackslashnrepeat (CAG) expansion in the coding region of the ATAXN2 gene onbackslashnchromosome 12,which produces an elongated,toxic polyglutamine tract,backslashnleading to Purkinje cell loss. There is currently no effective therapy.backslashnOne of the main obstacles that hampers therapeutic development is lackbackslashnof an ideal disease model. In this study,we have generated andbackslashncharacterized SCA2-induced pluripotent stem (iPS) cell lines as an inbackslashnvitro cell model. Dermal fibroblasts (FBs) were harvested from primarybackslashncultures of skin explants obtained from a SCA2 subject and a healthybackslashnsubject. For reprogramming,hOct4,hSox2,hKlf4,and hc-Myc werebackslashntransduced to passage-3 FBs by retroviral infection. Both SCA2 iPS andbackslashncontrol iPS cells were successfully generated and showed typical stembackslashncell growth patterns with normal karyotype. All iPS cell lines expressedbackslashnstem cell markers and differentiated in vitro into cells from threebackslashnembryonic germ layers. Upon in vitro neural differentiation,SCA2 iPSbackslashncells showed abnormality in neural rosette formation but successfullybackslashndifferentiated into neural stem cells (NSCs) and subsequent neuralbackslashncells. SCA2 and normal FBs showed a comparable level of ataxin-2backslashnexpression; whereas SCA2 NSCs showed less ataxin-2 expression thanbackslashnnormal NSCs and SCA2 FBs. Within the neural lineage,neurons had thebackslashnmost abundant expression of ataxin-2. Time-lapsed neural growth assaybackslashnindicated terminally differentiated SCA2 neural cells were short-livedbackslashncompared with control neural cells. The expanded CAG repeats of SCA2backslashnwere stable throughout reprogramming and neural differentiation. Inbackslashnconclusion,we have established the first disease-specific human SCA2backslashniPS cell line. These mutant iPS cells have the potential for neuralbackslashndifferentiation. These differentiated neural cells harboring mutationsbackslashnare invaluable for the study of SCA2 pathogenesis and therapeutic drugbackslashndevelopment. View Publication

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study,we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519CtextgreaterT (p.R120X) into induced pluripotent stem cells (iPSC),and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells,suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization,Golgi cohesion and G$\$1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren),we were able to restore up to 20% of endogenous,full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients. View Publication

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant protein-folding disorder caused by over 100 distinct mutations in the transthyretin (TTR) gene. In ATTR,protein secreted from the liver aggregates and forms fibrils in target organs,chiefly the heart and peripheral nervous system,highlighting the need for a model capable of recapitulating the multisystem complexity of this clinically variable disease. Here,we describe detailed methodologies for the directed differentiation of protein folding disease-specific iPSCs into hepatocytes that produce mutant protein,and neural-lineage cells often targeted in disease. Methodologies are also described for the construction of multisystem models and drug screening using iPSCs. View Publication

BACKGROUND: 1,25-Dihydroxyvitamin D(3) inhibits growth of several types of human cancer cells in vitro,but its therapeutic use is hampered because it causes hypercalcemia. 19-nor-1,25-Dihydroxyvitamin D(2) (paricalcitol) is a noncalcemic vitamin D analog that is approved by the Food and Drug Administration for the treatment of secondary hyperparathyroidism. We investigated the antitumor activity and mechanism of action of paricalcitol in vitro and in vivo. METHODS: Effects of paricalcitol on proliferation,the cell cycle,differentiation,and apoptosis were examined in cancer cell lines. Effects on tumor growth were examined with colon cancer cell xenografts in nude mice (five in the experimental group and five in the control group). The interaction of paricalcitol with the vitamin D receptor (VDR) in mononuclear spleen cells and myeloid stem cells from wild-type and VDR knockout mice was examined. All statistical tests were two-sided. RESULTS: Paricalcitol inhibited the proliferation of myeloid leukemia cell lines HL-60,NB-4,and THP-1 cells at an effective dose that inhibited growth 50% (ED(50)) of 2.4-5.8 x 10(-9) M by inducing cell cycle arrest and differentiation. Paricalcitol inhibited the proliferation of NCI-H929 myeloma cells at an ED(50) of 2.0 x 10(-10) M by inducing cell cycle arrest and apoptosis. Paricalcitol also inhibited the proliferation of colon cancer cell lines HT-29 (ED(50) = 1.7 x 10(-8) M) and SW837 (ED(50) = 3.2 x 10(-8) M). HT-29 colon cancer xenografts in paricalcitol-treated nude mice were smaller (1044 mm(3) and 1752 mm(3),difference = 708 mm(3),95% confidence interval = 311 to 1104 mm(3); P =.03) and weighed less (1487 mg and 4162 mg,difference = 2675 mg,95% confidence interval = 2103 to 3248 mg; Ptextless.001) than those in vehicle-treated mice. Paricalcitol induced committed myeloid hematopoietic stem cells from wild-type but not from VDR knockout mice to differentiate as macrophages. CONCLUSION: Paricalcitol has anticancer activity against myeloid leukemia,myeloma,and colon cancer cells that may be mediated through the VDR. Because it has been approved by the Food and Drug Administration,clinical trials of this agent in certain cancers are reasonable. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Cell transplantation has been well explored for cartilage regeneration. We recently showed that the entire articular surface of a synovial joint can regenerate by endogenous cell homing and without cell transplantation. However,the sources of endogenous cells that regenerate articular cartilage remain elusive. Here,we studied whether cytokines not only chemotactically recruit adipose stem cells (ASCs),mesenchymal stem cells (MSCs),and synovium stem cells (SSCs) but also induce chondrogenesis of the recruited cells. Recombinant human transforming growth factor-β3 (TGF-β3; 100 ng) and/or recombinant human stromal derived factor-1β (SDF-1β; 100 ng) was control released into an acellular collagen sponge cube with underlying ASCs,MSCs,or SSCs in monolayer culture. Although all cell types randomly migrated into the acellular collagen sponge cube,TGF-β3 and/or SDF-1β recruited significantly more cells than the cytokine-free control group. In 6 wk,TGF-β3 alone recruited substantial numbers of ASCs (558±65) and MSCs (302±52),whereas codelivery of TGF-β3 and SDF-1β was particularly chemotactic to SSCs (400±120). Proliferation of the recruited cells accounted for some,but far from all,of the observed cellularity. TGF-β3 and SDF-1β codelivery induced significantly higher aggrecan gene expression than the cytokine-free group for ASCs,MSCs,and SSCs. Type II collagen gene expression was also significantly higher for ASCs and SSCs by SDF-1 and TGF-β3 codelivery. Remarkably,the expression of aggrecan and type II collagen was detected among all cell types. Thus,homing of multiple stem/progenitor cell populations may potentially serve as an alternative or adjunctive approach to cell transplantation for cartilage regeneration. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication

Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency,it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells. 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