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Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells,but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors,we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation,reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast,sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression,decreased GATA1 expression,and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression,particularly its later decline,is key to producing functional platelets from selected iPSC clones. View Publication

BACKGROUND: Vascular trauma induced by surgical revascularization stimulates mobilization of hematopoietic and nonhematopoietic progenitor cells. However,it is not clear whether mobilized progenitors are functionally active and participate in peripheral homing. We have found no clinical studies available regarding the reaction of bone marrow to surgical revascularization. METHODS: This was an observational prospective study of 76 patients undergoing elective coronary artery bypass graft surgery. Bone marrow aspirates and blood samples were collected at baseline,at the end of surgery,and 24 hours postoperatively (blood samples only). The CD34+,CD34+CD133+,and CD34+CXCR4+ progenitor cell counts,CXCR4+ mononuclear cell counts,and CXCR4 expression on CD34+ cells were measured by flow cytometry. Progenitor cell functions were studied in vitro by clonogenic and migration assays. RESULTS: In response to coronary revascularization there was mobilization of CD34+ progenitors,having increased migratory and clonogenic function. The CD34+CXCR4+ subsets and CXCR4 expression on CD34+ cells in peripheral blood increased significantly 24 hours postoperatively. The CXCR4 expression on mobilized progenitors at the end of surgery was independently related to baseline CXCR4 expression on bone marrow resident CD34+ cells and duration of cardiopulmonary bypass in a multivariate model. At the end of surgery there was a significant fall in the expression of CXCR4 on CD34+ bone marrow cells,suggesting egress into peripheral circulation of the most active CXCR4-expressing progenitors. CONCLUSIONS: Coronary artery bypass graft surgery is associated with bone marrow release of functionally active progenitor cells. Further studies are needed to verify whether mobilized progenitors participate in regeneration of injured tissues. View Publication

BACKGROUND: Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor,thereby causing relapse and patient death. Ewing's sarcoma,the second most common form of bone tumor in adolescents and young adults,follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved,even for patients with metastatic disease,but relapse remains frequent and is usually fatal. METHODOLOGY/PRINCIPAL FINDINGS: We have isolated a subpopulation of Ewing's sarcoma cells,from both human cell lines and human xenografts grown in immune deficient mice,which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity,sphere-formation,and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro,but this can be overcome by the ATP binding cassette transport protein inhibitor,verapamil. Importantly,these cells are not resistant to YK-4-279,a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy. View Publication

Small molecules will need to be identified and/or developed that target protein classes limiting reprogramming efficiency. A specific class of proteins includes epigenetic regulators that silence,or minimize expression,of pluripotency genes in differentiated cells. To better understand the role of specific epigenetic modulators in reprogramming,we have used shRNA delivered by lentivirus to assess the significance of individual epi-proteins in reprogramming pluripotent gene expression. View Publication

BACKGROUND AIMS: Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS: We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation,and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS: The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P textless 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally,two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS: A combined staining of ALDH,CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets. View Publication

To exploit the full potential of human pluripotent stem cells for regenerative medicine,developmental biology and drug discovery,defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed,but finding chemically defined,robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides,which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. View Publication

The ability of hematopoietic stem cells (HSCs) to undergo self-renewal is partly regulated by external signals originating from the stem cell niche. Our previous studies with HSCs obtained from fetal liver of mice deficient for the calcium-sensing receptor (CaR) have shown the crucial role of this receptor in HSC lodgment and engraftment in the bone marrow (BM) endosteal niche. Using a CaR agonist,Cinacalcet,we assessed the effects of stimulating the CaR on the function of murine HSCs. Our results show that CaR stimulation increases primitive hematopoietic cell activity in vitro,including growth in stromal cell cocultures,adhesion to extracellular matrix molecules such as collagen I and fibronectin,and migration toward the chemotactic stimulus,stromal cell-derived factor 1α. Receptor stimulation also led to augmented in vivo homing,CXCR4-mediated lodgment at the endosteal niche,and engraftment capabilities. These mechanisms by which stimulating the CaR dictates preferential localization of HSCs in the BM endosteal niche provide additional insights into the fundamental interrelationship between the stem cell and its niche. These studies also have implications in the area of clinical stem cell transplantation,where ex vivo modulation of the CaR may be envisioned as a strategy to enhance HSC engraftment in the BM. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system,along with their G-coupled cannabinoid receptors (CB�? and CB₂) and the enzymes involved in their biosynthesis and degradation. However,their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here,we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol),whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists,CB₂ receptors induced chemotaxis,migration,and enhanced colony formation of bone marrow cells,which were mediated via ERK,PI3-kinase,and Gαi-Rac1 pathways. In vivo,the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition,granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together,these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus,CB₂ agonists may be therapeutically applied in clinical conditions,such as bone marrow transplantation. View Publication

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice,the development of granulocytes is favored at the expense of monocytes. However,Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras,we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils,Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα,C/EBPβ,and PU.1,depends on Btk. In addition,expression of several granule proteins,including myeloperoxidase,neutrophilic granule protein,gelatinase and neutrophil elastase,is Btk-dependent. In the Arthus reaction,an acute inflammatory response,neutrophil migration into tissues,edema formation,and hemorrhage are significantly reduced in Btk-deficient animals. Together,our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo. View Publication

Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently,it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self-renewal and pluripotency and,thus,bypassing senescence. Here,we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs,AFiPSCs,fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE (Lost,Acquired and Retained Gene Expression) Principle of Cellular Reprogramming,we could further highlight that AFiPSCs,FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4,SOX2 and NANOG. Nevertheless,these cell types are marked by distinct gene expression signatures. For example,expression of the transcription factors,SIX6,EGR2,PKNOX2,HOXD4,HOXD10,DLX5 and RAXL1,known to regulate developmental processes,are retained in AFiPSCs and FiPSCs. Surprisingly,expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this,with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs,warrant further studies. 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