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RATIONALE: Adults with cystic fibrosis (CF) are at increased risk of developing osteoporosis. During infective exacerbations,increased production of proinflammatory cytokines and markers of bone resorption have been reported. OBJECTIVE: The aim of this study is to investigate the growth and proliferation of potential osteoclast precursor cells before,during,and after intravenous antibiotic treatment of infective exacerbations in patients with CF. METHODS: Hematopoietic precursor cell growth was examined using colony formation assays using Methocult culture medium. Circulating potential osteoclast precursors were identified using four-color flow cytometry by CD14,CD33,CD34,and CD45 expression. RESULTS: At the start of an infective exacerbation increases in hematopoietic precursor colony formation (15.42 colonies/10(5) cells plated,p = 0.025),proliferation (28.5%,p textless 0.001),and the numbers of circulating potential osteoclast precursors (6.5%,p textless 0.001) were seen in comparison with baseline levels. These increases declined after treatment with intravenous antibiotics to a level close to baseline. CONCLUSIONS: The results demonstrate an increase in the production of potential osteoclast precursors in the peripheral blood during CF infective exacerbations. This may result in increased bone resorption and contribute to bone loss in patients with CF. View Publication

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently,a new pluripotent population of umbilical cord blood (UCB) cells,unrestricted somatic stem cells (USSCs),with intrinsic and directable potential to develop into mesodermal,endodermal,and ectodermal fates,has been identified. In this study,we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold,respectively; p textless .05),with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells,as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation,with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells,generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p textless .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. View Publication

The HOXA9 homeoprotein exerts dramatic effects in hematopoiesis. Enforced expression of HOXA9 enhances proliferation of primitive blood cells,expands hematopoietic stem cells (HSCs),and leads to myeloid leukemia. Conversely,loss of HOXA9 inhibits proliferation and impairs HSC function. The pathways by which HOXA9 acts are largely unknown,and although HOXA9 is a transcription factor,few direct target genes have been identified. Our previous study suggested that HOXA9 positively regulates Pim1,an oncogenic kinase. The hematologic phenotypes of Hoxa9- and Pim1-deficient animals are strikingly similar. Here we show that HOXA9 protein binds to the Pim1 promoter and induces Pim1 mRNA and protein in hematopoietic cells. Pim1 protein is diminished in Hoxa9(-/-) cells,and Hoxa9 and Pim1 mRNA levels track together in early hematopoietic compartments. Induction of Pim1 protein by HOXA9 increases the phosphorylation and inactivation of the proapoptotic BAD protein,a target of Pim1. Hoxa9(-/-) cells show increased apoptosis and decreased proliferation,defects that are ameliorated by reintroduction of Pim1. Thus Pim1 appears to be a direct transcriptional target of HOXA9 and a mediator of its antiapoptotic and proproliferative effects in early cells. Since HOXA9 is frequently up-regulated in acute myeloid leukemia,Pim1 may be a therapeutic target in human disease. View Publication

The erythroid response to acute anemia relies on the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress BFU-E. This expansion requires BMP4/Madh5-dependent signaling in vivo; however,in vitro,BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo,which suggests that other signals are required. In this report we show that mutation of the Kit receptor results in a severe defect in the expansion of stress BFU-E,indicating a role for the Kit/SCF signaling pathway in stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E,but only when spleen cells were cultured in BMP4 + SCF at low-oxygen concentrations did we recapitulate the expansion of stress BFU-E observed in vivo. Culturing spleen cells in BMP4,SCF under hypoxic conditions resulted in the preferential expansion of erythroid progenitors characterized by the expression of Kit,CD71,and TER119. This expression pattern is also seen in stress erythroid progenitors isolated from patients with sickle cell anemia and patients with beta-thalassemia. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia. View Publication

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations,many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity,cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly,the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development,which are highly sensitive to Runx1 dosage. However,RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated,showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly,both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro,accompanied by the accumulation of myeloblasts and dysplastic progenitors,but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta,an important cofactor of Runx1,did not impair RDB mutant replating activity,arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling. View Publication

The T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of the Jak/Stat cytokine signaling pathway. Our study shows that the absence of TC-PTP leads to an early bone marrow B-cell deficiency characterized by hindered transition from the pre-B cell to immature B-cell stage. This phenotype is intrinsic to the B cells but most importantly due to bone marrow stroma abnormalities. We found that bone marrow stromal cells from TC-PTP(-/-) mice have the unique property of secreting 232-890 pg/mL IFN-gamma. These high levels of IFN-gamma result in 2-fold reduction in mitotic index on IL-7 stimulation of TC-PTP(-/-) pre-B cells and lower responsiveness of IL-7 receptor downstream Jak/Stat signaling molecules. Moreover,we noted constitutive phosphorylation of Stat1 in those pre-B cells and demonstrated that this was due to soluble IFN-gamma secreted by TC-PTP(-/-) bone marrow stromal cells. Interestingly,culturing murine early pre-B leukemic cells within a TC-PTP-deficient bone marrow stroma environment leads to a 40% increase in apoptosis in these malignant cells. Our results unraveled a new role for TC-PTP in normal B lymphopoiesis and suggest that modulation of bone marrow microenvironment is a potential therapeutic approach for selected B-cell leukemia. View Publication

OBJECTIVE: In vitro models of hematopoiesis used in investigative hematopathology and in safety studies on candidate drugs,involve clonogenic assays on colony-forming unit granulocyte macrophage (CFU-GM). These assays require live and unstained colonies to be counted. Most laboratories still rely on visual scoring,which is time-consuming and error-prone. As a consequence,automated scoring is highly desired. An algorithm that recognizes and scores CFU-GM colonies by data fusion has been developed. Some preliminary results are presented in this article. METHODS: CFU-GM assays were carried out on hematopoietic progenitors (human umbilical cord blood cells) grown in methylcellulose. Colony images were acquired by a digital camera and stored. RESULTS: The classifier was designed to process images of layers sampled from a three-dimensional (3D) domain and forming a stack. Structure and texture information was extracted from each image. Classifier training was based on a 3D colony model applied to the image stack. The number of scored colonies (assigned class) was required to match the count supplied by the human expert (class of belonging). The trained classifier was validated on one more stack and then applied to a stack with overlapping colonies. Scoring in distortion- and caustic-affected border areas was also successfully demonstrated. Because of hardware limitations,compact colonies in some cases were missed. CONCLUSIONS: The industry's scoring methods all rely on structure alone and process 2D data. Instead,the classifier here fuses data from a whole stack and is capable,in principle,of high-throughput screening. View Publication

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported,and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes,CD31 and CD144,in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells,freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes,marginal upregulation of CD31,and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however,no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters,which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage. View Publication

The effect of sustained exposure to nicotine,a major constituent of cigarette smoke,on hematopoiesis in the bone marrow (BM) and spleen was evaluated in a murine model. BALB/c mice were exposed to nicotine subcutaneously using 21-day slow-release pellets. Exposure to nicotine had no effect on the proliferation of long-term BM cultures or on their ability to form colonies. However,there was a significant decrease in the generation of lineage-specific progenitor cells,specifically eosinophil (colony-forming unit [CFU]-Eos) progenitors,in the BM of nicotine-exposed mice compared with control mice. Surprisingly,sustained exposure of mice to nicotine was found to induce significant hematopoiesis in the spleen. There was a significant increase in total colony formation as well as eosinophil-,granulocyte-macrophage-,and B-lymphocyte-specific progenitors (CFU-Eos,CFU-GM,and CFU-B,respectively) in nicotine-exposed mice but not in control mice. Sustained exposure to nicotine was associated with significant inhibition of rolling and migration of enriched hematopoietic stem/progenitor cells (HSPCs) across BM endothelial cells (BMECs) in vitro as well as decreased expression of beta2 integrin on the surface of these cells. Although sustained exposure to nicotine has only a modest effect on BM hematopoiesis,our studies indicate that it significantly induces extramedullary hematopoiesis in the spleen. Decreased interaction of nicotine-exposed HSPCs with BMECs (i.e.,rolling and migration) may result in altered BM homing of these cells,leading to their seeding and proliferation at extramedullary sites such as the spleen. View Publication

Supplementation of mesenchymal stem cells (MSCs) during hematopoietic stem cell (HSC) transplantation alleviates complications such as graft-versus-host disease,leading to a speedy recovery of hematopoiesis. To meet this clinical demand,a fast MSC expansion method is required. In the present study,we examined the feasibility of using a rotary bioreactor system to expand MSCs from isolated bone marrow mononuclear cells. The cells were cultured in a rotary bioreactor with Myelocult medium containing a combination of supplementary factors,including stem cell factor and interleukin-3 and -6. After 8 days of culture,total cell numbers,Stro-1(+)CD44(+)CD34(-) MSCs,and CD34(+)CD44(+)Stro-1(-) HSCs were increased 9-,29-,and 8-fold,respectively. Colony-forming efficiency-fibroblast per day of the bioreactor-treated cells was 1.44-fold higher than that of the cells without bioreactor treatment. The bioreactor-expanded MSCs showed expression of primitive MSC markers endoglin (SH2) and vimentin,whereas markers associated with lineage differentiation,including osteocalcin (osteogenesis),type II collagen (chondrogenesis),and C/EBP-alpha (CCAAT/enhancer-binding protein-alpha) (adipogenesis),were not detected. Upon induction,the bioreactor-expanded MSCs were able to differentiate into osteoblasts,chondrocytes,and adipocytes. We conclude that the rotary bioreactor with the modified Myelocult medium reported in this study may be used to rapidly expand MSCs. View Publication

Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps,similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently,CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99,albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid,natural killer (NK),or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors. View Publication

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH),hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon,we investigated the usefulness of ALDH activity for characterizing HSC graft quality,particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time,and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly,in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells,this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism,whereas all other patients showed early complete donor chimerism. In conclusion,we suggest to measure ALDH activity as a surrogate marker for HSC activity,and to transport and store PBSC under controlled cooling conditions. View Publication