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BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47,CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47,higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression,arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated,in part,by increased CD44-cytoskeleton binding. View Publication

Bone marrow injury is a major adverse side effect of radiation and chemotherapy. Attempts to limit such damage are warranted,but their success requires a better understanding of how radiation and anticancer drugs harm the bone marrow. Here,we report one pivotal role of the BH3-only protein Puma in the radiosensitivity of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). Puma deficiency in mice confers resistance to high-dose radiation in a hematopoietic cell-autonomous manner. Unexpectedly,loss of one Puma allele is sufficient to confer mice radioresistance. Interestingly,null mutation in Puma protects both primitive and differentiated hematopoietic cells from damage caused by low-dose radiation but selectively protects HSCs and HPCs against high-dose radiation,thereby accelerating hematopoietic regeneration. Consistent with these findings,Puma is required for radiation-induced apoptosis in HSCs and HPCs,and Puma is selectively induced by irradiation in primitive hematopoietic cells,and this induction is impaired in Puma-heterozygous cells. Together,our data indicate that selective targeting of p53 downstream apoptotic targets may represent a novel strategy to protecting HSCs and HPCs in patients undergoing intensive cancer radiotherapy and chemotherapy. View Publication

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients,we now demonstrate that some,but not all,of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P textless .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders,P textless .003). In contrast,CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly,one BCR-ABL mutation (V304D),predicted to destabilize the interaction between p210(BCR-ABL) and IM,was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus,2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management. View Publication

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment,we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy,c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages,whereas erythroid differentiation was impaired,as demonstrated by clonogenic assay,morphologic and immunophenotypic data. Gene expression profiling and computational analysis of promoter regions of genes modulated in c-myb-silenced CD34(+) cells identified the transcription factors Kruppel-Like Factor 1 (KLF1) and LIM Domain Only 2 (LMO2) as putative targets,which can account for c-myb knockdown effects. Indeed,chromatin immunoprecipitation and luciferase reporter assay demonstrated that c-myb binds to KLF1 and LMO2 promoters and transactivates their expression. Consistently,the retroviral vector-mediated overexpression of either KLF1 or LMO2 partially rescued the defect in erythropoiesis caused by c-myb silencing,whereas only KLF1 was also able to repress the megakaryocyte differentiation enhanced in Myb-silenced CD34(+) cells. Our data collectively demonstrate that c-myb plays a pivotal role in human primary hematopoietic stem/progenitor cells lineage commitment,by enhancing erythropoiesis at the expense of megakaryocyte diffentiation. Indeed,we identified KLF1 and LMO2 transactivation as the molecular mechanism underlying Myb-driven erythroid versus megakaryocyte cell fate decision. View Publication

This study aimed to determine the mechanisms responsible for long-term tissue damage following radiation injury. We irradiated p21-knockout (p21(-/-)) and wild-type (WT) mice and determined the long-term deleterious effects of this intervention on mesenchyme-derived tissues. In addition,we explored the mechanisms of radiation-induced mesenchymal stem cell (MSC) dysfunction in isolated bone marrow-derived cells. p21 expression was chronically elevated textgreater200-fold in irradiated tissues. Loss of p21 function resulted in a textgreater4-fold increase in the number of skin MSCs remaining after radiation. p21(-/-) mice had significantly less radiation damage,including 6-fold less scarring,40% increased growth potential,and 4-fold more hypertrophic chondrocytes in the epiphyseal plate (Ptextless0.01). Irradiated p21(-/-) MSCs had 4-fold increased potential for bone or fat differentiation,4-fold greater proliferation rate,and nearly 7-fold lower senescence as compared to WT MSCs (Ptextless0.01). Ectopic expression of p21 in knockout cells decreased proliferation and differentiation potential and recapitulated the WT phenotype. Loss of p21 function markedly decreases the deleterious effects of radiation injury in mesenchyme-derived tissues and preserves tissue-derived MSCs. In addition,p21 is a critical regulator of MSC proliferation,differentiation,and senescence both at baseline and in response to radiation. View Publication

TOX is a DNA-binding factor required for development of CD4(+) T cells,natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells,a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis,required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow,consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus,many cell lineages of the immune system share a TOX-dependent step for development. View Publication

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure,we found that Fancd2(-/-) mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit(+)Sca-1(+)Lineage(-) (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2(-/-) KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition,the supportive function of the marrow microenvironment was compromised in Fancd2(-/-) mice. Treatment with Sirt1-mimetic and the antioxidant drug,resveratrol,maintained Fancd2(-/-) KSL cells in quiescence,improved the marrow microenvironment,partially corrected the abnormal cell cycle status,and significantly improved the spleen colony-forming capacity of Fancd2(-/-) bone marrow cells. We conclude that Fancd2(-/-) mice have readily quantifiable hematopoietic defects,and that this model is well suited for pharmacologic screening studies. View Publication

CONTEXT: The osteoanabolic properties of PTH may be due to increases in the number and maturity of circulating osteogenic cells. Hypoparathyroidism is a useful clinical model because this hypothesis can be tested by administering PTH. OBJECTIVE: The objective of the study was to characterize circulating osteogenic cells in hypoparathyroid subjects during 12 months of PTH (1-84) administration. DESIGN: Osteogenic cells were characterized using flow cytometry and antibodies against osteocalcin,an osteoblast-specific protein product,and stem cell markers CD34 and CD146. Changes in bone formation from biochemical markers and quadruple-labeled transiliac crest bone biopsies (0 and 3 month time points) were correlated with measurements of circulating osteogenic cells. SETTING: The study was conducted at a clinical research center. PATIENTS: Nineteen control and 19 hypoparathyroid patients were included in the study. INTERVENTION: Intervention included the administration of PTH (1-84). RESULTS: Osteocalcin-positive cells were lower in hypoparathyroid subjects than controls (0.7 ± 0.1 vs. 2.0 ± 0.1%; P textless 0.0001),with greater coexpression of the early cell markers CD34 and CD146 among the osteocalcin-positive cells in the hypoparathyroid subjects (11.0 ± 1.0 vs. 5.6 ± 0.7%; P textless 0.001). With PTH (1-84) administration,the number of osteogenic cells increased 3-fold (P textless 0.0001),whereas the coexpression of the early cell markers CD34 and CD146 decreased. Increases in osteogenic cells correlated with circulating and histomorphometric indices of osteoblast function: N-terminal propeptide of type I procollagen (R(2) = 0.4,P ≤ 0.001),bone-specific alkaline phosphatase (R(2) = 0.3,P textless 0.001),osteocalcin (R(2) = 0.4,P textless 0.001),mineralized perimeter (R(2) = 0.5,P textless 0.001),mineral apposition rate (R(2) = 0.4,P = 0.003),and bone formation rate (R(2) = 0.5,P textless 0.001). CONCLUSIONS: It is likely that PTH stimulates bone formation by stimulating osteoblast development and maturation. Correlations between circulating osteogenic cells and histomorphometric indices of bone formation establish that osteoblast activity is being identified by this methodology. 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

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation,potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here,we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF,containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically,UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally,XF and SF-cultured UCMSC have comparable adipogenic,osteogenic,and endothelial differentiation potential. Interestingly,the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression,transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation,and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together,GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine. View Publication

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase,leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However,because a suitable hematopoietic donor is not found for everyone,because HCT is associated with significant morbidity and mortality,and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH,gene-corrected autologous stem cells may be the ideal graft for HCT. Thus,we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal,and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation,as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT. View Publication

Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. Based on these properties of self-renewal and pluripotency,hESCs represent a unique source to generate a large quantity of certain specialized cell types with clinical interest for transplantation-based therapy. However,hESCs are usually grown in culture conditions using fetal bovine serum and mouse embryonic fibroblasts,two components that are not compatible with clinical applications. Consequently,the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here,we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders. View Publication