Effect of tresperimus on in vitro human cord blood CD34+ cell differentiation

Tresperimus, an analogue of 15-deoxyspergualin (15-DSG), is immunosuppressive and prevents lethal graft-versus-host disease following allogeneic bone marrow transplantation in mice. Here, we present an in vitro dose response study examining the ability of tresperimus to support clonogenesis in cultured CD34+ cord blood stem cells. Our findings revealed that only the lowest dose examined, 0.5 microg tresperimus/ml, supports normal myelopoiesis, erythropoiesis and megakaryopoiesis. Greater concentrations of the drug induced dose-dependent inhibition of clonogenesis. This latter effect was not due to apoptosis and was reversible by drug withdrawal. We conclude that tresperimus at 0.5 microg/ml supports the clonogenic potential of cord blood CD34+ cells. Dose-dependent inhibition of clonogenesis was completely reversible following drug withdrawal. These results may be of clinical interest as tresperimus is currently used in phase I-III studies for the prevention of graft versus host disease in recipients of allogeneic bone marrow.     

Production of human B cells from CD34+CD38- T- B- progenitors in organ culture by sequential cytokine stimulation

We investigated sequential cytokine addition on human hematopoietic stem cell (HSC) differentiation in murine fetal liver (FL), fetal spleen (FS) and bone marrow (BM) organ cultures (OC). Tissues were colonized with unpurified or FACS sorted CD34+CD38-CD10-CD19-CD3-CD8-CD4-(T- B-) cells from human cord blood (HUCB). CD19+ cell production and kinetics differed in each tissue. Fetal liver organ cultures (FLOC) inoculated with CD34+CD38-T-B- cells produced fewer CD19+ cells than fetal liver organ culture (FLOC) cultured with unpurified HUCB. CD19+ cell production was restored in the CD34+CD38-T-B- organ cultures by treating with SCF, LIF and IL-6 followed by IL-7 and removing all cytokines for the last 3 days of culture (a six-fold increase). FLOC also produced CD34+CD38-T-B- cells and monocyte-lineage CD33+CD14- cells, both of which increased after cytokine treatment. Re-colonization of secondary FLOC with CD34+CD38-T-B- cells generated in primary FLOC produced additional B-cells, monocytes and CD34+CD38- cells suggesting that the primary cells retained HSC activity. Expansion and differentiation of HSCs depended on the microenvironment of the recipient tissue as well as addition of cytokines in the appropriate order.     

Correlation between IL-3 receptor expression and growth potential of human CD34+ hematopoietic cells from different tissues.

CD123 (alpha-subunit of IL-3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single-cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34+ cells were defined by the levels of surface CD123: CD123negative, CD123low, and CD123bright. Coexpression of lineage markers showed that a majority of CD34+CD123bright cells were myeloid and B-lymphoid progenitors, while erythroid progenitors were mainly in the CD34+CD123negative subset. The CD34+CD123low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34+ cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell factor, interleukin 3 (IL-3), IL-6, GM-CSF, erythropoietin, insulin-like growth factor-1, and basic fibroblast growth factor. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34+CD123negative and CD34+CD123low fractions (37+/-23% and 44+/-23%, respectively) than in the CD34+CD123bright fraction (15+/-21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed growth pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34+CD123negative and CD34+CD123low subsets. When CD38 and CD90 (Thy-1) were used for further characterization of CD34+ cells from FLV, CE increased from 37+/-23% in CD123negative to 70+/-19% in CD123negativeCD38- and from 44+/-23% in CD123low to 66+/-19% in CD123lowCD38-. No significant increase in CE or DGP progenitors was observed when CD34+ cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B-lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123negative/low expression.     

Characterization of a lineage-negative stem-progenitor cell population optimized for ex vivo expansion and enriched for LTC-IC

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long-term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long-term in vivo hematopoiesis. We report here an optimized, rapid negative-isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage-negative (Lin-) cell population (0.10% +/- 0.02% MNC, n=12). This primitive Lin- cell population encompassed CD34+/- and CD133+/- HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin- cells being highly enriched for long-term culture-initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c-Kit ligand 20 ng/ml) Lin- cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin- stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well-characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.     

Defining optimum conditions for the ex vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels

Ex vivo expansion of human umbilical cord blood cells (HUCBC) is explored by several investigators to enhance the repopulating potential of HUCBC. We performed experiments using either Ficoll-separated or CD34+-selected HUCBC from the same donation in serum-free medium. CD34-purified HUCBC were cultured on either human umbilical vein endothelial cells (HUVEC) or irradiated bone marrow-derived stroma cells (BMSC) with addition of different cytokines. In addition, we tested the expansion of HUCBC in culture vessels with continuous rotation. CD34 enrichment led to a significant increase in the expansion factor of CD34+ cells compared with unmanipulated HUCBC. BMSC were more efficient in amplifying early progenitors than HUVEC. Optimum results were reached by a combination of SCF, FLT-3L at 300 ng/ml and IL-3 at 50 ng/ml. No significant improvement in the expansion of CD34+/38- primitive progenitors could be obtained with other combinations. Addition of megakaryocyte-derived growth and development factor to each growth factor cocktail improved the expansion results. Continuous rotation of culture vessels did not ameliorate the expansion rate of the analyzed subsets. Culture conditions separating stroma and HUCBC by a semipermeable membrane improved the expansion factors of CD34+, CD34+/38-, and CD34+/41+ cells and CFU-GM compared with contact cultures. These data might be useful when designing culture systems for clinical scale ex vivo expansion of HUCBC.     

Cycling G1 CD34+/CD38+ cells potentiate the motility and engraftment of quiescent G0 CD34+/CD38-/low severe combined immunodeficiency repopulating cells

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34(+)-enriched cells induces the production of short-term repopulating, cycling G1 CD34(+)/CD38(+) cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell-derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF-1-mediated in vitro migration and in vivo homing of quiescent G0 CD34(+) cells, which is partially abrogated after inhibition of MMP-2/-9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34(+)/CD38(+) cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti-CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34(+)/CD38(+) committed progenitor cells and quiescent, primitive G0 CD34(+)/CD38(-/low) SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP-9.     

骨髓间充质干细胞对骨髓CD34~+细胞增殖影响的体外研究

研究骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMMSC)对骨髓CD34+细胞增殖的影响,为骨髓CD34+细胞体外扩增的深入研究提供依据。用MACS进行骨髓CD34+细胞的分选;用流式细胞术进行CD34+细胞纯度鉴定;用密度梯度离心法分离引产胎儿骨髓单个核细胞,结合贴壁法进行BMMSC的体外扩增培养;用流式细胞术进行BMMSC表面标志鉴定;用Transwell培养板培养CD34+细胞与BMMSC;用自动细胞计数仪计数有活性的CD34+细胞数量;用MTT比色法检测骨髓CD34+细胞增殖活性。流式细胞术检测结果显示分选所得的CD34+细胞纯度达到90%以上;流式细胞术检测显示传至第3代继续培养72h后的BMMSC高表达CD44、CD29,而HLA-DR、CD45、CD34表达阴性,说明所培养的BMMSC纯度很高;Transwell培养板培养CD34+细胞与BMMSC,在倒置显微镜下观察以及自动细胞计数仪计数发现,实验组有活性的CD34+细胞数量高于对照组(P<0.05);采用MTT比色法检测骨髓CD34+细胞的增殖活性,实验组高于对照组(P<0.05)。以上结果说明BMMSC有促进骨髓CD34+细胞增殖的作用,这种作用可能与其分泌的细胞因子有关。     

Adipocyte differentiation of human marrow mesenchymal stem cells reduces the supporting capacity for hematopoietic progenitors but not for severe combined immunodeficiency repopulating cells

Bone marrow stromal cells provide a microenvironment for hematopoiesis. Adipocytes are the major stromal cell phenotype in bone marrow, but their function in hematopoiesis is poorly understood. In this study, we compared the hematopoietic-supporting capacity of adipocytes and their progenitor, mesenchymal stem cells (MSCs), by culturing human cord blood (CB) CD34+CD38- hematopoietic progenitor cells (HPCs) on a layer of adipocytes or MSCs. CB CD34+CD38- cells cultured on MSCs generated higher proportions of CD34+CD38- HPCs and colony-forming cells than those cultures on a layer of adipocytes, indicating an inferior hematopoietic support by adipocytes. However, CB CD34+CD38- HPCs cultured on MSCs and adipocytes were equally capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings show that differentiation of MSCs into adipocytes is accompanied by the loss of capacity to support mature HPCs, but not transplantable SCID-repopulating cells.     

Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells.

So far, blood progenitor cells (BPC) expanded ex vivo in the absence of stromal cells have not been demonstrated to reconstitute hematopoiesis in myeloablated patients. To characterize the fate of early hematopoietic progenitor cells during ex vivo expansion in suspension culture, human CD34(+)-enriched BPC were cultured in serum-free medium in the presence of FLT3 ligand (FL), stem cell factor (SCF) and interleukin 3 (IL-3). Both CD34 surface expression levels and the percentage of CD34+ cells were continuously downregulated during the culture period. We observed an expansion of colony-forming units granulocyte-macrophage (CFU-GM) and BFU-E beginning on day 3 of culture, reaching an approximate 2-log increase by days 5 to 7. Limiting dilution analysis of primitive in vitro clonogenic progenitors was performed through a week 6 cobblestone-area-forming cell (CAFC) assay, which has previously been shown to detect long-term bone marrow culture-initiating cells (LTC-IC). A maintenance or a slight (threefold) increase of week 6 CAFC/LTC-IC was found after one week of culture. To analyze the presence of BPC mediating in vivo engraftment, expanded CD34+ cells were transplanted into preirradiated NOD/SCID mice at various time points. Only CD34+ cells cultured for up to four days successfully engrafted murine bone marrow with human cells expressing myeloid or lymphoid progenitor phenotypes. In contrast, five- and seven-day expanded human BPC did not detectably engraft NOD/SCID mice. When FL, SCF and IL-3-supplemented cultures were performed for seven days on fibronectin-coated plastic, or when IL-3 was replaced by thrombopoietin, colony forming cells and LTC-IC reached levels similar to those of control cultures, yet no human cell engraftment was recorded in the mice. Also, culture in U-bottom microplates resulting in locally increased CD34+ cell density had no positive effect on engraftment. These results indicate that during ex vivo expansion of human CD34+ cells, CFC and LTC-IC numbers do not correlate with the potential to repopulate NOD/SCID mice. Our results suggest that ex vivo expanded BPC should be cultured for limited time periods only, in order to preserve bone-marrow-repopulating hematopoietic stem cells.     

Fetal bovine bone marrow is a rich source of CD34+ hematopoietic progenitors with myelo-monocytic colony-forming activity

The CD34 glycoprotein is an important marker of hematopoietic stem cells. We used a polyclonal rabbit anti-bovine CD34 antibody to stain fetal and adult bovine bone marrow cells. Flow cytometry revealed a low side scatter (SSC^low) population of cells that were CD34⁺ but negative for leukocyte lineage markers CD11b, CD14 or CD2. Hematopoietic colony assays with CD34⁺ and CD34⁻ bone marrow cells suggested that the colony-forming potential in SSC^low bone marrow cells was confined to the CD34⁺ fraction. In contrast, this population was not enriched for cells expressing high aldehyde dehydrogenase activity, a metabolic marker that has been used to characterize hematopoietic stem cells. Thus, the CD34 antigen can be used to identify and isolate bovine bone marrow cells exhibiting clonogenic potential in vitro. Moreover, the proportion of CD34⁺ cells is very high in fetal bovine bone marrow, indicating it as a rich source of hematopoietic progenitors.     

Bone marrow CD34+ progenitor cells stimulated with stem cell factor and GM-CSF have the capacity to activate IgD- B cells through direct cellular interaction

Recent studies have suggested the involvement of bone marrow in the pathogenesis of rheumatoid arthritis (RA), in which proliferation of monocyte-lineage cells (MLC) as well as local B cell activation in the synovium play an important role. Here, we show that bone marrow-derived MLC have the capacity to activate human peripheral blood IgD- B cells. Bone marrow CD34+ cells from RA patients that had been stimulated with stem cell factor and GM-CSF for 3-4 weeks (>90% CD14+ HLA-DR+ cells, <0.5% CD19+ B cells, and <0.5% CD3+ T cells; MLC) induced the production of IgG much more effectively than that of IgM by highly purified B cells from healthy donors in the presence of IL-2 and IL-10. CD34+ cells from cord blood or from bone marrow of osteoarthritis patients also displayed the capacity to induce IgG production. The induction of IgG production by the bone marrow-derived MLC was markedly decreased when they were separated from B cells by a membrane filter. The bone marrow-derived MLC interacted preferentially with IgD- B cells to induce IgG production. These results indicate that upon stimulation with stem cell factor and GM-CSF, CD34+ progenitor cells differentiate into MLC that activate preferentially IgD- B cells through direct cellular interactions to produce IgG. Therefore, the data suggest that the accelerated recruitment of MLC from the bone marrow to the synovium might play a role in the local B cell activation in RA.     

Catecholaminergic neurotransmitters regulate migration and repopulation of immature human CD34+ cells through Wnt signaling

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we report that immature human CD34+ cells dynamically expressed dopamine and beta2-adrenergic receptors, with higher expression in the primitive CD34+CD38(lo) population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through Wnt signaling activation and increased cell mobilization and bone marrow Sca-1+c-Kit+Lin- cell numbers. Our results identify new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration and development.     

Identification and hematopoietic potential of CD45- clonal cells with very immature phenotype (CD45-CD34-CD38-Lin-) in patients with myelodysplastic syndromes

CD45 is a hematopoietic lineage-restricted antigen that is expressed on all hematopoietic cells except for some mature cell types. Cells expressing CD45 and CD34 but lacking CD38 and lineage antigens (CD45+CD34+CD38-Lin- cells) are well-documented hematopoietic stem cells (HSCs), and CD45+CD34-CD38-Lin- cells are probably less mature HSCs. In myelodysplastic syndromes (MDS), the malignant transformation site is a matter of debate, and CD45+CD34+CD38-Lin- HSCs were recently reported to be clonal. In the study reported here, we detected CD45-CD34-CD38-Lin- cells in the peripheral blood and bone marrow of patients with MDS and isolated them by successive application of density centrifugation, magnetic cell sorting, and fluorescence-activated cell sorting. Fluorescence in situ hybridization showed that CD45-CD34-CD38-Lin- cells had the same chromosomal aberration as the myeloblasts. In addition to CD45- and CD34-, they lacked CD117 and CD133 expression. Generally, MDS cells have extremely reduced hematopoietic potential compared with normal hematopoietic cells, but we documented the following in some patients. Freshly isolated CD45-CD34-CD38-Lin- cells did not form any hematopoietic colonies but had long-term culture-initiating cell activity. When cocultured with stroma cells, CD45-CD34-CD38-Lin- cells showed only weak potential for proliferation and differentiation, yet they differentiated into CD34+ cells and then mature myeloid cells. This newly identified cell population represents the most immature immunophenotype so far identified in the hematopoietic lineage and is involved in the malignant clone in MDS.     

Cytokine-induced expansion of human CD34+ stem/progenitor and CD34+CD41+ early megakaryocytic marrow cells cultured on normal osteoblasts

Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.     

Colocalization analysis of sialomucins CD34 and CD164

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34(+) cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.     

Gene-expression profiling of CD34+ hematopoietic cells expanded in a collagen I matrix

CD34+ hematopoietic stem/progenitor cells (HSCs) reside in the bone marrow in close proximity to the endosteal bone surface, surrounded by osteoblasts, stromal cells, and various extracellular matrix molecules. We used a bioartificial matrix of fibrillar collagen I, the major matrix component of bone, as a scaffold for ex vivo expansion of HSCs. CD34+ HSCs were isolated from umbilical cord blood and cultivated within reconstituted collagen I fibrils in the presence of fms-like tyrosine kinase-3 ligand, stem cell factor, and interleukin (IL)-3. After 7 days of culture, the cell number, number of colony-forming units (CFU-C), and gene-expression profile of the cultured cells were assessed. Although the total expansion factor of CD34+ cells was slightly lower when cells were cultivated in the collagen I gel, the frequency of CFU-C was greater than in control suspension cultures. Gene-expression analysis with microarray chip technology revealed the upregulation of more than 50 genes in the presence of collagen I. Among these, genes for several growth factors, cytokines, and chemokines (e.g., IL-8 and macrophage inhibitory protein 1alpha) could be confirmed using quantitative polymerase chain reaction. Furthermore, greater expression levels of the negative cell-cycle regulator BTG2/TIS21 and an inhibitor of the mitogen-activated protein kinase pathway, DUSP2, underline the regulatory role of the extracellular matrix. Together, these data show that the expansion of CD34+ cord blood cells in a culture system containing a three-dimensional collagen I matrix induces a qualitative change in the gene-expression profile of cultivated HSCs.     

Induction of fibroblast-like cells from CD34(+) progenitor cells of the bone marrow in rheumatoid arthritis

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34(+) cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34(+) cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha), much more effectively than CD34(+) cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-alpha. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-alpha-stimulated cultures of bone marrow CD34(+) cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34(+) cells from patients with RA have abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34(+) progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.     

Murine stromal cell line HESS-5 maintains reconstituting ability of Ex vivo-generated hematopoietic stem cells from human bone marrow and cytokine-mobilized peripheral blood

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units, and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture, the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality.     

Idiopathic CD4+ lymphocytopenia may be due to decreased bone marrow clonogenic capability

BACKGROUND: Idiopathic CD4+ lymphocytopenia is defined by a stable decrease of CD4+ T cells in the absence of any known cause of immune deficiency. The mechanisms responsible for the immunological impairment are still unknown, but a regenerative failure of hematopoietic stem/progenitor cells has been hypothesized. METHODS: We evaluated in the bone marrow (BM) of 5 patients with idiopathic CD4+ lymphocytopenia the phenotype of BM progenitor cells, their differentiation capacity with colony-forming cells and long-term culture-initiating cell assays, in parallel with the spontaneous IL-7 production in the patient sera. RESULTS: Compared with controls, a regenerative failure of hematopoietic stem cells has been observed, both in 'committed' and in 'uncommitted' progenitor cells, despite high IL-7 serum levels. The percentage of phenotypically primitive CD34+CD38-DR+ cells (this includes the lymphoid precursor cells) was decreased, suggesting an involvement of the more primitive BM compartment in the de novo T cell generation. CONCLUSIONS: Despite the low number of patients, due to the low incidence of the disease, the decrease of primitive precursors sustains the possibility that diminished stem cell precursors might contribute to the development of CD4+ T cell depletion.