CD34high+ CD38(low/-) cells generated in a xenogenic coculture system are capable of both long-term hematopoiesis and multiple differentiation.

CD34+ cells isolated from human umbilical cord blood (HUCB) are thought to have potential in clinical applications such as transplantation and gene therapy. Recently, we developed a xenogenic coculture system involving HUCB-CD34+ cells and murine bone marrow stromal cells, HESS-5 cells, in combination with human interleukin-3 and stem cell factor. Under these xenogenic coculture conditions, the numbers of CD34high+ cells and primitive progenitor cells, such as CD34high+ CD38(low/-) cells and high proliferative potential colony-forming cells (HPP-CFCs), increased dramatically by a factor of 102.1, 66.5 and 104.9, respectively. In the present study, we used a secondary culture of B progenitor cells and long-term culture (LTC)-initiating cells to characterize and compare the progenitor capability of re-isolated CD34high+ CD38(low/-) cells, which have been identified as one of the most primitive progenitor cells, with that of freshly isolated CD34high+ CD38(low/-) cells. Compared with freshly isolated CD34high+ CD38(low/-) cells, the re-isolated CD34high+ CD38(low/-) cells were equally as capable of proliferating and differentiating into myeloid and B progenitor cells. No significant differences were observed in the frequency of LTC-initiating cells in the re-isolated CD34high+ CD38(low/-) cells compared with that in freshly isolated CD34high+ CD38(low/-) cells. Furthermore, the re-isolated CD34high+ CD38(low/-) cells were capable of long-term reconstitution and multiple differentiation in non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). The results demonstrate that this xenogenic coculture system can be used for successful in vitro expansion of HUCB-progenitor cells that possess the capability for both long-term hematopoiesis as well as multipotent differentiation into myeloid and lymphoid cells both in vivo and in vitro.     

Human cord blood long-term engrafting cells are CD34+ CD38-.

There have been controversies about CD34 and CD38 expression by human cord blood (CB) stem cells. Using the newborn NOD/SCID/beta2-microglobulin-null mouse assay that we recently developed, we examined the in vivo engrafting capability of human CB cells. Almost all of the 4-5 months engrafting cells were found in CD34(+) population. The capability of secondary reconstitution was found only in the CD34(+) cells. When the CD34(+) CB cells were separated into CD38(-) and CD38(+) subpopulations and tested for engraftment, the majority of the engrafting cells were detected in the CD38(-) subpopulation. These findings are consistent with the results from studies of murine stem cells and strongly indicate that the phenotype of human CB stem cells is CD34(+) CD38(-).     

Selectively frequent expression of CXCR5 enhances resistance to apoptosis in CD8(+)CD34(+) T cells from patients with T-cell-lineage acute lymphocytic leukemia

We investigated CD4(+)CD34(+), CD8(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells from cord blood and from typical patients with T-cell-lineage acute lymphocytic leukemia and T-cell-lineage chronic lymphocytic leukemia in terms of expression and functions of CXCR5/CXCL13. We found that CXCR5 was selectively frequently expressed on T-cell-lineage acute (chronic) lymphocytic leukemia (T-ALL) CD8(+)CD34(+) T cells, but not on T-ALL CD4(+)CD34(+), CD4(+)CD34(-), and CD8(+)CD34(-) T cells. CXCR5 was rarely expressed on all types of CD34(+) and CD34(-) CB or T-CLL T cells. CXCL13/B cells attracting chemokine 1 induced significant resistance to TNF-alpha-mediated apoptosis in T-ALL CD8(+)CD34(+) T cells, instead of induction of chemotactic and adhesive responsiveness. A proliferation-inducing ligand expression in T-ALL CD8(+)CD34(+) T cells was upregulated by CXCL13/BCA-1 (B-cell attracting chemokine 1). The CXCR5/CXCL13 pair by means of activation of APRIL (A proliferation-inducing ligand) induced resistance to apoptosis in T-ALL CD8(+)CD34(+) T cells in livin-dependent manner. In this process, cell-cell contact in culture was necessary. Based on our findings, we suggested that there were differential functions of CXCR5/CXCL13 in distinct types of cells. Normal lymphocytes, especially naive B and T cells, utilized CXCR5/CXCL13 for migration, homing, maturation, and cell homeostasis, as well as secondary lymphoid tissue organogenesis. Meanwhile, certain malignant cells took advantages of CXCR5/CXCL13 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

Retinoic acid induction of CD38 antigen expression on normal and leukemic human myeloid cells: relationship with cell differentiation

Differentiation in the hematopoietic system involves, among other changes, altered expression of antigens, including the CD34 and CD38 surface antigens. In normal hematopoiesis, the most immature stem cells have the CD34+CD34 -phenotype. In acute myeloid leukemia (AML), although blasts from most patients are CD38+, some are CD38 -. AML blasts are blocked at early stages of differentiation; in some leukemic cells this block can be overcome by a variety of agents, including retinoids, that induce maturation into macrophages and granulocytes both in vitro and in vivo . Retinoids can also induce CD38 expression. In the present study, we investigated the relationship between induction of CD38 expression and induction of myeloid differentiation by retinoic acid (RA) in normal and leukemic human hematopoietic cells. In the promyelocytic (PML) CD34 -cell lines, HL60 and CB-1, as well as in normal CD34+CD34 -hematopietic progenitor cells RA induced both CD38 expression as well as morphological and functional myeloid differentiation that resulted in loss of self-renewal. In contrast, in the myeloblastic CD34+ leukemic cell lines, ML-1 and KG-1a, as well as in primary cultures of cells derived from CD34+-AML (M 0 and M 1 ) patients, RA caused an increase in CD38+ that was not associated with significant differentiation. Yet, long exposure of ML-1, but not KG-1, cells to RA resulted in loss of self-renewal. The results suggest that while in normal hematopoietic cells and in PML CD34 -cells induction of CD38 antigen expression by RA results in terminal differentiation along the myeloid lineage, in early myeloblastic leukemic CD34+ cells, induction of CD38 and differentiation are not functionally related. Since, several lines of evidence suggest that the CD38 -cells are the targets of leukemic transformation, transition of these cells into CD38+ phenotype by RA or other drugs may have therapeutic effect, either alone or in conjunction with cytotoxic drugs, regardless the ability of the cells to undergo differentiation.     

Functional alterations of Lin-CD34+CD38+ cells in chronic myelomonocytic leukemia and on progression to acute leukemia

The functional behavior of hematopoietic stem cell (HSC) and progenitors in chronic myelomonocytic leukemia (CMML) and on disease progression is little known. We performed cell proliferation, apoptosis, hematopoietic colony forming/replating and differentiation potential studies in the purified subpopulations of Lin(-)CD34(+)CD38(-) and Lin(-)CD34(+)CD38(+) cells from 16 CMML with 6 cases after acute myeloid leukemia transformation (AML-t). We observed an expansion of the hematopoietic progenitor pool (Lin(-)CD34(+) cells) in AML-t comprising mainly Lin(-)CD34(+)CD38(+) cells. The Lin(-)CD34(+)CD38(+) cells in AML-t displayed high proliferative activity, resistance to apoptosis, enhanced myeloid colony formation/replating ability and a complete dendritic cell (DC) differentiation block. Our findings suggest Lin(-)CD34(+)CD38(+) cells instead of Lin(-)CD34(+)CD38(-) cells could be the target(s) of secondary genetic lesions underpinning progression from CMML to AML, which have implications for the further study of the biology of leukemic transformation and the design of new strategies for the effective treatment of CMML.     

Development of human lymphohematopoiesis defined by CD34 and CD81 expression

Human blood cells, except for erythrocytes and platelets, express CD81, a member of the transmembrane 4 superfamily (TM4SF). CD81 is also expressed on most of human immature hematopoietic cells, CD34+ cells, which are divided into three populations according to the expression of CD34 and CD81; CD34+CD81+, CD34+CD81(High) and CD34(Low)CD81+. Myeloid and lymphoid progenitors exist in the CD34+CD81+ population, and megakaryocytic progenitors are only in CD34(Low)CD81+ population. Erythroid and multipotential progenitors are shared by CD34+CD81+ and CD34(Low)CD81+ populations, but multipotential progenitors in the CD34+CD81+ population have already lost most of their myeloid potential. NK cells and mast cells can be generated from all three populations. Long-term repopulating (LTR) lymphohematopoietic stem cells are present in the CD34+CD81+ population. Based on these findings, we propose a model for the development of CD34+CD81+ lymphohematopoietic stem cells. Along the differentiation cascade from CD34+CD81+ lymphohematopoietic stem cells, there appear to be pathways to CD34(Low)CD81 + or CD34+CD81(High) cells, even if they are indirect. CD34(Low)CD81+ pathways define the loss of LTR ability, and lymphoid and myeloid potentials, whereas CD34+CD81(High) pathways represent the exclusive commitment to NK cells and mast cells.     

CD34(+) or CD34(-): which is the more primitive?

Remarkable progress has been achieved in the characterization and isolation of primitive hematopoietic stem cells (HSC). HSC represent a very small subset of hematopoietic cells and provide self-renewal, possess differentiation capacity and allow a constant supply of the entire hematopoietic cell spectrum. Until recently, CD34 has been used as a convenient marker for HSC, since CD34(+) cells have been shown to possess colony-forming potential in short-term assays, maintain long-term colony-forming potential in in vitro cultures and allow the expression and differentiation of blood cells from different hematopoietic lineages in in vivo models. Clinical and experimental protocols have targeted CD34(+) cells enriched by a variety of selection models and have readily used these for transplantation, purging and gene therapies and targets for future organ replacement. Recent studies in murine and human models, however, have indicated that CD34(-) HSC exist as well, which possess engraftment potential and distinct HSC characteristics. These studies challenge the dogma that HSC are uniformly found in the CD34(+) subset, and question whether primitive HSC are CD34(+) or CD34(-). In this review, results on murine and human CD34(+) and CD34(-) HSC, differences between them and their possible interactions are examined.     

Flow cytometric detection of aneuploid CD38(++) plasmacells and CD19(+) B-lymphocytes in bone marrow, peripheral blood and PBSC harvest in multiple myeloma patients

DNA aneuploidy has been used as a genetic marker of malignancy in multiple myeloma (MM). CD38 and CD138 expression and absence of CD22 and CD19 may define plasmacells (PC). Several authors support evidences of circulating plasmacells, and their role in relapse after autologous stem cell transplantation has been hypothesised. The existence of B-lymphocytes belonging to the myeloma clone is still controversial. If CD19 or CD22 positive B-lymphocytes are part of the myeloma clone, there should be evidence of myeloma-specific genetic markers in this population. Using DNA content measurement in combination with CD19 or CD38 detection in a multiparametric flow cytometry analysis, we studied bone marrow and peripheral blood of 10 aneuploid MM patients. In the bone marrows of all these 10 aneuploid patients (100%), we detected CD38(++) aneuploid plasmacells ( 27 +/- 17%, mean +/- S.D.) and a small number of CD19(+) aneuploid lymphocytes ( 0.11 +/- 0.074%). In 100% of these patients, we also detected CD38(++) aneuploid circulating plasmacells ( 0.6 +/- 0.9 %) and a small number of CD19(+) aneuploid lymphocytes (0.03 +/- 0.04%). In this study, we detected aneuploid CD19(+) lymphocytes and CD38(++) plasmacells in bone marrow and peripheral blood of all MM patients. A crucial role for the detection of aneuploid CD19(+) cells was played by the acquisition of a sufficient number of CD19(+) lymphocytes by using a "live gate" acquisition and "continuous gating" analysis. With the techniques used in this study, it was possible to detect aneuploid B lymphoid cells among normal diploid B cells. The significance of this finding is controversial and opened to different interpretations.     

Notch ligand Delta-1 differentially modulates the effects of gp130 activation on interleukin-6 receptor alpha-positive and -negative human hematopoietic progenitors

Interleukin (IL)-6 plays pleiotropic roles in human hematopoiesis and immune responses by acting on not only the IL-6 receptor-alpha subunit (IL-6Ralpha)(+) but also IL-6Ralpha(-) hematopoietic progenitors via soluble IL-6R. The Notch ligand Delta-1 has been identified as an important modulator of the differentiation and proliferation of human hematopoietic progenitors. Here, it was investigated whether these actions of IL-6 are influenced by Delta-1. When CD34(+)CD38(-) hematopoietic progenitors were cultured with stem cell factor, flt3 ligand, thrombopoietin and IL-3, Delta-1, in combination with the IL-6R/IL-6 fusion protein FP6, increased the generation of glycophorin A(+) erythroid cells but counteracted the effects of IL-6 and FP6 on the generation of CD14(+) monocytic and CD15(+) granulocytic cells. Although freshly isolated CD34(+)CD38(-) cells expressed no or only low levels of IL-6Ralpha, its expression was increased in myeloid progenitors after culture but remained negative in erythroid progenitors. It was found that Delta-1 acted in synergy with FP6 to enhance the generation of erythroid cells from the IL-6Ralpha(-) erythroid progenitors. In contrast, Delta-1 antagonized the effects of IL-6 and FP6 on the development of monocytic and granulocytic cells, as well as CD14(-)CD1a(+) dendritic cells, from the IL-6Ralpha(+) myeloid progenitors. These results indicate that Delta-1 interacts differentially with gp130 activation in IL-6Ralpha(-) erythroid and IL-6Ralpha(+) myeloid progenitors. The present data suggest a divergent interaction between Delta-1 and gp130 activation in human hematopoiesis.     

乳腺癌肿瘤干细胞标记物CD44^＋ESA^＋CD24^∧ow在非小细胞肺癌组织中的表达及其意义

背景与目的：人体各种组织都存在干细胞,肿瘤组织也存在肿瘤干细胞（tumor stem cell,TSC）。乳腺癌TSC已经被分离出来,其标记物也已被确定,但肺癌的TSC仍未被分离出来。本研究旨在探讨乳腺癌肿瘤干细胞标记物（CD44^＋ESA^＋CD24^∧ow）在非小细胞肺癌（NSCLC）组织中的表达及其意义。方法：应用免疫组织化学法检测77例NSCLC组织中CD44、ESA与CD24的表达,分析其与患者吸烟、肿瘤的大小、癌的组织学类型、组织分化程度、淋巴结转移和预后的关系。结果：77例NSCLC组织中CD44、ESA与CD24的阳性率分别为63．6％、66．2％和7,8％。低分化及未分化组CD44的阳性率明显高于高分化组,高分化组ESA的阳性率明显高于中度分化组和低分化及未分化组;腺癌组ESA的阳性率明显高于鳞癌组（P〈0．05）。CD44^＋ESA^＋CD24^∧ow标记的阳性率为36．4％,与患者吸烟、肿瘤的大小、癌的组织学类型、组织分化程度、淋巴结转移和预后无关（P〉0．05）。结论：乳腺癌肿瘤干细胞标记物（CD44^＋ESA^＋CD24^∧ow）表达与NSCLC肿瘤的大小、癌的组织学类型、组织分化程度、淋巴结转移和预后等临床病理学指标无关。     

The effect of tetraethylenepentamine, a synthetic copper chelating polyamine, on expression of CD34 and CD38 antigens on normal and leukemic hematopoietic cells

We have previously found that the synthetic polyamine tetraethylenepentamine (TEPA) significantly delayed differentiation and prolonged expansion of cord-blood derived HPC in cytokine-supplemented cultures. Most HPC have the CD34+CD38+ phenotype, but the minority CD34+38- cells are primitive subset of HPC that have the potential for long-term repopulation in vivo. We investigated the effect of TEPA on the CD34/CD38 surface antigen expression of human myeloid leukemia cell lines as well as normal cord blood derived hematopoietic cells. Confirming previous results, our data showed that both the leukemic and normal cells increased their CD38 expression when grown in serum-containing medium or when treated with retinoic acid. In the present study, we found that TEPA inhibited CD38 under these conditions in both normal and leukemic cells. As for CD34, TEPA increased the proportion of CD34 cells in short- and long-term normal cultures but not in the leukemic cell lines. These results suggest that ex vivo expansion of HPC depends on the presence of CD34+CD38- cells and that TEPA prolongs HPC expansion by inhibiting the CD38- to CD38+ transition.     

Severe functional alterations in vitro in CD34(+) cell subpopulations from patients with chronic myeloid leukemia

Chronic myeloid leukemia (CML) arises from the malignant transformation of a hematopoietic stem cell (HSC) that gives rise to functionally defective progeny, including primitive and relatively mature progenitor cells (HPC). Both HSC and HPC are comprised within the population of CD34(+) cells, normally present in bone marrow (BM). In the present study, we have separated two different subpopulations of CD34(+) cells from CML marrow: Population I, enriched for CD34(+) Lin(-) cells; and Population II, enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells, and assessed their progenitor cell content as well as their capacity to proliferate and expand in response to a combination of hematopoietic cytokines in serum- and stroma-free long-term liquid cultures. The absolute cell numbers recovered in Population I from normal and CML samples were similar; in contrast, we found that Population II from CML was amplified four-fold, as compared to normal. In spite of this latter observation, no significant differences were observed in terms of the absolute number of CFC when comparing Populations I and II from CML patients and normal subjects. Interestingly, the proliferation and expansion potentials of CML cells were clearly deficient as compared to their normal counterparts. Indeed, in cultures of Population I cells the maximum fold increase in total and progenitor cell numbers corresponded to 30 and 8%, respectively, of those observed in cultures of normal marrow-derived Population I cells. Such functional deficiencies were even more evident in Population II cells in which the maximum fold increase in total and progenitor cell numbers corresponded to 3 and 0.5%, respectively, of the levels found in cultures of Population II cells from normal marrow. The present study demonstrates that bone marrow-derived CD34(+) cells from CML patients possess functional abnormalities, clearly evident in the in vitro system used by us. Among the two CML subpopulations studied here, the more immature one (Population II; enriched for CD34(+) CD36(-) CD38(-) CD45RA(-) Lin(-) cells) was the one that showed the most severe abnormalities, as compared to its relatively more mature counterpart (Population I; enriched for CD34(+) Lin(-) cells).     

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.     

Distinct molecular phenotype of malignant CD34(+) hematopoietic stem and progenitor cells in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a malignant disorder of the hematopoietic stem cell characterized by the BCR-ABL oncogene. We examined gene expression profiles of highly enriched CD34(+) hematopoietic stem and progenitor cells from patients with CML in chronic phase using cDNA arrays covering 1.185 genes. Comparing CML CD34(+) cells with normal CD34(+) cells, we found 158 genes which were significantly differentially expressed. Gene expression patterns reflected BCR-ABL-induced functional alterations such as increased cell-cycle and proteasome activity. Detoxification enzymes and DNA repair proteins were downregulated in CML CD34(+) cells, which might contribute to genetic instability. Decreased expression of junction plakoglobulin and CXC chemokine receptor 4 (CXCR-4) might facilitate the release of immature precursors from bone marrow in CML. GATA-2 was upregulated in CML CD34(+) cells, suggesting an increased self-renewal in comparison with normal CD34(+) cells. Moreover, we found upregulation of the proto-oncogene SKI and of receptors for neuromediators such as opioid mu1 receptor, GABA B receptor, adenosine A1 receptor, orexin 1 and 2 receptors and corticotropine-releasing hormone receptor. Treatment of CML progenitor cells with the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) resulted in a dose-dependent significant inhibition of clonogenic growth by 40% at a concentration of 10(-5) M, which could be reversed by the equimolar addition of the receptor agonist 2-chloro-N6-cyclopentyladenosine (P<0.05). The incubation of normal progenitor cells with DPCPX resulted in an inhibition of clonogenic growth to a significantly lesser extent in comparison with CML cells (P<0.05), suggesting that the adenosine A1 receptor is of functional relevance in CML hematopoietic progenitor cells.     

Comparative multi-color flow cytometric analysis of cell surface antigens in bone marrow hematopoietic progenitors between refractory anemia and aplastic anemia

Refractory anemia (RA) in myelodysplastic syndrome (MDS) without prominent dysplasia closely resemble the mild type of aplastic anemia (AA) in their hematological features. This sometimes makes it difficult to distinguish clearly between the two diseases. Using the multi-color flow cytometric technique, we compared cell surface antigen expression patterns on bone marrow hematopoietic progenitor cells which were isolated as a CD34 positive- CD45 dull positive with low side scatter intensity (CD34(+)CD45(dull+)SSC(low)) population in flow cytogram between RA (n=12) and AA (n=11). The antigens analyzed in CD34(+)CD45(dull+)SSC(low) mononuclear cells were: CD38 and CD71 for cell growth-related antigens, CD 33 and CD13 for myeloid and monocytoid lineage-associated antigens, CD7 and CD19 for lymphoid lineage, and CD14 for a monocytic lineage specific antigen. The percentages of CD34(+)CD45(dull+)SSC(low) cells in bone marrow non-erythroid mononuclear cells, and the expression frequencies of CD38, CD71, CD33 and CD13 antigens in CD34(+)CD45(dull+)SSC(low) progenitors were all significantly decreased in AA compared to normal bone marrows (n=7) (P<0.005). In contrast, in RA bone marrows the percentages of CD34(+)CD45(dull+)SSC(low) cells showed wide distribution and the cell surface antigen expression patterns varied among each case: some cases showed low frequencies of CD38 and CD71 expression as well as AA, whereas the others showed high expression frequency of specific antigen(s) which may reflect the clonal expansion of an abnormal clone in bone marrow. An MDS patient who had progressed from RA to RAEB showed further projecting pattern of expression of CD38 and CD33 in CD34(+)CD45(dull+)SSC(low) population in accordance with the disease progression. These data suggest that analysis of cell surface antigen expression patterns of CD34(+)CD45(dull+)SSC(low) progenitor cells by multi-color flow cytometry appears to be a useful method for qualitative and quantitative assessment of marrow progenitor states in AA and RA, therefore this method could be helpful for early detection of clonal evolution in MDS.     

Retinoic acid receptor antagonist inhibits CD38 antigen expression on human hematopoietic cells in vitro

The CD34+ CD38- subset of human hematopoietic stem cells are crucial for long-term ex-vivo expansion; conditions that decreased this specific sub-population reduced the self-renewal capacity and shortened the duration of the proliferative phase of the culture. Retinoids, such as all-trans retinoic acid (ATRA), have been shown to induce CD38 expression. ATRA present in serum may be responsible for the high CD38 of cells grown in serum-containing medium. In the present study we analyzed the effects of AGN 194310, a retinoic acid receptor pan-antagonist, on CD38 expression of human hematopoietic cells. Normal cells (cord blood derived CD34+ cells) and abnormal cells (myeloid leukemic lines) were studied when grown in either serum-containing or serum-free media. The results showed that both serum and ATRA enhanced differentiation and, thereby, reduced the proportion of CD34+ CD38- cells and total CD34+ cell expansion. AGN reversed these effects of serum and ATRA: it delayed differentiation and increased CD34+ CD38- cells. These results suggest that physiological ATRA levels in serum may prevent efficient cell expansion. AGN, by neutralizing ATRA, improves cell expansion in serum-containing cultures, thus making AGN a useful agent for ex vivo expansion of stem cells and other specific sub-populations for research and clinical use.     

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.     

Proangiogenic factor PlGF programs CD11b(+) myelomonocytes in breast cancer during differentiation of their hematopoietic progenitors

Tumor-mobilized bone marrow-derived CD11b(+) myeloid cells promote tumor angiogenesis, but how and when these cells acquire proangiogenic properties is not fully elucidated. Here, we show that CD11b(+) myelomonocytic cells develop proangiogenic properties during their differentiation from CD34(+) hematopoietic progenitors and that placenta growth factor (PlGF) is critical in promoting this education. Cultures of human CD34(+) progenitors supplemented with conditioned medium from breast cancer cell lines or PlGF, but not from nontumorigenic breast epithelial lines, generate CD11b(+) cells capable of inducing endothelial cell sprouting in vitro and angiogenesis in vivo. An anti-Flt-1 mAb or soluble Flt-1 abolished the generation of proangiogenic activity during differentiation from progenitor cells. Moreover, inhibition of metalloproteinase activity, but not VEGF, during the endothelial sprouting assay blocked sprouting induced by these proangiogenic CD11b(+) myelomonocytes. In a mouse model of breast cancer, circulating CD11b(+) cells were proangiogenic in the sprouting assays. Silencing of PlGF in tumor cells prevented the generation of proangiogenic activity in circulating CD11b(+) cells, inhibited tumor blood flow, and slowed tumor growth. Peripheral blood of breast cancer patients at diagnosis, but not of healthy individuals, contained elevated levels of PlGF and circulating proangiogenic CD11b(+) myelomonocytes. Taken together, our results show that cancer cells can program proangiogenic activity in CD11b(+) myelomonocytes during differentiation of their progenitor cells in a PlGF-dependent manner. These findings impact breast cancer biology, detection, and treatment.