Characterization of murine CD34, a marker for hematopoietic progenitor and stem cells

CD34 is expressed on human hematopoietic stem and progenitor cells, and its clinical usefulness for the purification of stem cells has been well established. However, a similar pattern of expression for murine CD34 (mCD34) has not yet been determined. Two polyclonal anti-mCD34 antibodies that specifically recognize both endogenous and recombinant murine CD34 were developed to characterize the mCD34 protein and to determine its pattern of expression on murine cell lines and hematopoietic progenitor cells. Fluorescence-activated cell sorter analysis showed that mCD34 is expressed on NIH/3T3 embryonic fibroblasts, PA6 stromal cells, embryonic stem cells, M1 leukemia cells, and a subpopulation of normal bone marrow cells. Murine CD34 was found to be a glycoprotein expressed on the cell surface as either a full-length (approximately 100 kD) or truncated (approximately 90 kD) protein in NIH/3T3 and PA6 cells. Recombinant full-length CD34, when expressed in the CHO-K1 cell line, had a molecular weight of approximately 105 kD. Full-length CD34 expressed on M1 leukemia cells, had a higher apparent molecular weight (110 kD). These results suggest that there are glycosylation differences between CD34 expressed by different cell types. The full-length form, but not the truncated form, is a phosphoprotein that is hyperphosphorylated in response to 12-0- Tetradecanoyl phorbol 13-acetate treatment, suggesting potential functional differences between the two forms. Selection of the 3% highest-expressing CD34+ bone marrow cells enriched for the hematopoietic precursors that form colony-forming unit-spleen (CFU-S), CFU-granulocyte-macrophage, and burst-forming unit-erythroid. Transplantation of lethally irradiated mice with these cells demonstrated both short- and long-term repopulating ability, indicating that this population contains both functional hematopoietic progenitors and the putative stem cell. These antibodies should be useful to select for murine hematopoietic stem cells.     

Hematopoietic defects in mice lacking the sialomucin CD34

Although the pluripotent hematopoietic stem cell can only be definitively identified by its ability to reconstitute the various mature blood lineages, a diversity of cell surface antigens have also been specifically recognized on this subset of hematopoietic progenitors. One such stem cell-associated antigen is the sialomucin CD34, a highly O-glycosylated cell surface glycoprotein that has also been shown to be expressed on all vascular endothelial cells throughout murine embryogenesis as well as in the adult. The functional significance of CD34 expression on hematopoietic progenitor cells and developing blood vessels is unknown. To analyze the involvement of CD34 in hematopoiesis, we have produced both embryonic stem (ES) cells and mice that are null for the expression of this mucin. Analysis of yolk saclike hematopoietic development in embryoid bodies derived from CD34- null ES cells showed a significant delay in both erythroid and myeloid differentiation that could be reversed by transfection of the mutant ES cells with CD34 constructs expressing either a complete or truncated cytoplasmic domain. Measurements of colony-forming activity of hematopoietic progenitor cells derived from yolk sacs or fetal livers isolated from CD34-null embryos also showed a decreased number of these precursor cells. In spite of these diminished embryonic hematopoietic progenitor numbers, the CD34-null mice developed normally, and the hematopoietic profile of adult blood appeared typical. However, the colony-forming activity of hematopoietic progenitors derived from both bone marrow and spleen is significantly reduced in adult CD34-deficient animals, and these CD34-deficient progenitors also appear to be unable to expand in liquid cultures in response to hematopoietic growth factors. Even with these apparent progenitor cell deficiencies, CD34- null animals showed kinetics of erythroid, myeloid, and platelet recovery after sublethal irradiation that are indistinguishable from wild-type mice. These data strongly suggest that CD34 plays an important role in the formation of progenitor cells during both embryonic and adult hematopoiesis. However, the hematopoietic sites of adult CD34-deficient mice may still have a significant reservoir of progenitor cells that allows for normal recovery after nonmyeloablative peripheral cell depletion.     

Primitive hematopoietic cells in murine bone marrow express the CD34 antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin- /10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.     

Canine CD34: cloning of the cDNA and evaluation of an antiserum to recombinant protein

Increasingly, enriched populations of hematopoietic progenitors are used in experimental and clinical transplantation studies. The separation of progenitors is based on the expression of CD34, a marker preferentially expressed on progenitor cells. The dog model has been important for preclinical transplant studies, because it has proven predictive for outcomes in human hematopoietic stem cell transplantation. To identify and isolate canine hematopoietic progenitors, we have cloned a cDNA encoding a CD34 homologue from a canine myelomonocytic leukemia cell line, ML2. The CD34 homologue cDNA predicts an amino acid sequence that is highly conserved with human and murine CD34 in the cytoplasmic domain, transmembrane domain, and C- terminal end of the extracellular domain, but shows considerable divergence from these sequences at the amino-terminal end of the protein. In Western blotting studies, canine CD34 homologue (caCD34) appears to be a heavily and variably glycosylated protein with a molecular weight of approximately 100 kD and shows some tissue-specific differences in protein mass. To evaluate the expression of caCD34 protein, the extracellular domain of caCD34 was expressed as an Ig fusion protein and used as an immunogen to generate a rabbit polyclonal antiserum. The antiserum reacted against the fusion protein, against vascular endothelium, and with three leukemic cell lines. Approximately 1% of canine bone marrow cells stained brightly with antibodies to caCD34 and this population was 25- to 50-fold enriched for colony- forming units-granulocyte-macrophage as compared to unfractionated marrow mononuclear cells. These findings suggest that the canine CD34 homologue is expressed on bone marrow progenitor cells and, thus, that this molecule should be a valuable marker for identifying and isolating canine hematopoietic progenitors for experimental hematopoiesis and stem cell transplantation.     

MicroRNA-29a and microRNA-142-3p are regulators of myeloid differentiation and acute myeloid leukemia

Although microRNAs (miRNAs) are increasingly linked to various physiologic processes, including hematopoiesis, their function in the myeloid development is poorly understood. We detected up-regulation of miR-29a and miR-142-3p during myeloid differentiation in leukemia cell lines and CD34(+) hematopoietic stem/progenitor cells. By gain-of-function and loss-of-function experiments, we demonstrated that both miRNAs promote the phorbol 12-myristate 13-acetate-induced monocytic and all-trans-retinoic acid-induced granulocytic differentiation of HL-60, THP-1, or NB4 cells. Both the miRNAs directly inhibited cyclin T2 gene, preventing the release of hypophosphorylated retinoblastoma and resulting in induction of monocytic differentiation. In addition, a target of miR-29a, cyclin-dependent kinase 6 gene, and a target of miR-142-3p, TGF-β-activated kinase 1/MAP3K7 binding protein 2 gene, are involved in the regulation of both monocytic and granulocytic differentiation. A significant decrease of miR-29a and 142-3p levels and an obvious increase in their target protein levels were also observed in blasts from acute myeloid leukemia. By lentivirus-mediated gene transfer, we demonstrated that enforced expression of either miR-29a or miR-142-3p in hematopoietic stem/progenitor cells from healthy controls and acute myeloid leukemia patients down-regulated expression of their targets and promoted myeloid differentiation. These findings confirm that miR-29a and miR-142-3p are key regulators of normal myeloid differentiation and their reduced expression is involved in acute myeloid leukemia development.     

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.     

The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting- gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.     

CD34+ endothelial cell lines derived from murine yolk sac induce the proliferation and differentiation of yolk sac CD34+ hematopoietic progenitors

Embryonic hematopoiesis is initiated in part in the blood islands of the yolk sac. Previous confocal microscopic analysis has shown that the CD34 antigen, a mucin-like cell surface glycoprotein that is expressed by hematopoietic progenitors and all endothelial cells of the adult and embryo, is also found on a subset of luminal hematopoietic-like cells in the yolk sac blood islands as well as on the vascular endothelium lining these early hematopoietic locations. We show here that, as in all other hematopoietic sites thus far examined, immunoaffinity- purified CD34+ nonadherent cells from murine yolk sacs contain the vast majority of erythroid and myeloid progenitor cell colony forming activity. To examine the developmental interactions between these CD34+ hematopoietic progenitor cells of the yolk sac and the CD34+ yolk sac endothelium, we have immunaffinity-purified adherent endothelial cells from day 10.5 yolk sacs using CD34 antiserum and produced cell lines by transformation with a retrovirus expressing the polyoma middle T antigen. Analysis of these cell lines for CD34, von Willebrand's factor, FLK 1 and FLT 1 expression, and capillary growth in Matrigel indicates that they appear to be endothelial cells, consistent with their original phenotype in vivo. Coculture of yolk sac CD34+ hematopoietic cells on these endothelial cell lines results in up to a 60-fold increase in total hematopoietic cell number after approximately 8 days. Analysis of these expanded hematopoietic cells showed that the majority were of the monocyte/macrophage lineage. In addition, examination of the cultures showed the rapid formation of numerous cobblestone areas, a previously described morphologic entity thought to be representative of early pluripotential stem cells. Scrutiny of the ability of these endothelial cell lines to expand committed progenitor cells showed up to a sixfold increase in erythroid and myeloid colony- forming cells after 3 to 6 days in culture, consistent with the notion that these embryonic endothelial cells mediate the expansion of these precursor cells. Polymerase chain reaction analyses showed that most of the cell lines produce FLK-2/FLT-3 ligand, stem cell factor, macrophage colony-stimulating factor, leukemia-inhibitory factor, and interleukin- 6 (IL-6), whereas there is a generally low or not measurable production of granulocyte colony-stimulating factor, granulocyte-macrophage colony- stimulating factor, IL-1, IL-3, transforming growth factor beta-1, erythropoietin, or thrombopoietin. The output of mature hematopoietic cells from these cocultures can be modified to include an erythroid population by the addition of exogenous erythropoietin. These data suggest that endothelial cell lines derived form the yolk sac provide an appropriate hematopoietic environment for the expansion and differentiation of yolk sac progenitor cells into at least the myeloid and erythroid lineages.     

Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging

DNA methylation plays an important role in the self-renewal of hematopoietic stem cells and in the commitment to the lymphoid or myeloid lineages. Using purified CD34? hematopoietic progenitor cells and differentiated myeloid cell populations from the same human samples, we obtained detailed methylation profiles at distinct stages of hematopoiesis. We identified a defined set of differentiation-related genes that are methylated in CD34? hematopoietic progenitor cells but show pronounced DNA hypomethylation in monocytes and in granulocytes. In addition, by comparing hematopoietic progenitor cells from umbilical cord blood to hematopoietic progenitor cells from peripheral blood of adult donors we were also able to analyze age-related methylation changes in CD34? cells. Interestingly, the methylation changes observed in older progenitor cells showed a bimodal pattern with hypomethylation of differentiation-associated genes and de novo methylation events resembling epigenetic mutations. Our results thus provide detailed insight into the methylation dynamics during differentiation and suggest that epigenetic changes contribute to hematopoietic progenitor cell aging.     

Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome

The BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.     

Integrin engagement-induced inhibition of human myelopoiesis is mediated by proline-rich tyrosine kinase 2 gene products

OBJECTIVE: Hematopoietic progenitor proliferation and differentiation are inhibited by integrin engagement of fibronectin (FN). Focal adhesion kinases have been shown to mediate intracellular signaling from integrins, and we recently demonstrated that gene expression and pre-mRNA splicing of the focal adhesion kinase, PYK2, is abnormal in CD34(+) cells from chronic myelogenous leukemia (CML) patients. Here we investigated whether PYK2 gene products mediate integrin signaling in hematopoietic stem and progenitor cells. METHODS: Cord blood CD34(+) cells were retrovirally transduced with vectors encoding Pyk2H, Pyk2, or the dominant negative-acting, kinase-deficient, C-terminal PYK2 fragment, PRNK, and myeloid proliferation and differentiation was assessed using colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and liquid culture assays. RESULTS: CD34(+) cells overexpressing Pyk2H or Pyk2 generated 50% less colony-forming unit granulocyte/macrophage (CFU-GM) than eGFP-transduced controls. Although the number of CFC generated by PRNK-expressing cells was unchanged, LTC-IC were significantly reduced. Culture of CD34(+) cells on FN significantly reduced the generation of mature myeloid cells vs those cultured on BSA-coated wells, and could be overcome by addition of SCF. As is observed when integrins are engaged, overexpression of either Pyk2H or Pyk2 decreased committed myeloid progenitor proliferation and differentiation; however, SCF could not override this inhibition. Finally, as is observed when integrins are not engaged, PRNK-mediated inhibition of endogenous Pyk2H resulted in integrin-nonresponsive proliferation and differentiation of myeloid precursors and accelerated differentiation of primitive hematopoietic progenitors. CONCLUSION: These studies indicate that PYK2 gene products mediate integrin-induced signals that regulate myelopoiesis.     

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34⁺ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34⁺ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34⁺ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34⁺ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.     

Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.     

Human hematopoiesis in murine embryos after injecting human cord blood-derived hematopoietic stem cells into murine blastocysts.

At different developmental stages, candidate human hematopoietic stem cells (HSCs) are present within the CD34+ CD38- population. By means of xenotransplantation, such CD34+CD38- cells were recently shown to engraft the hematopoietic system of fetal sheep and nonobese diabetic severe combined immunodeficient adult mice. Here it is demonstrated that, after their injection into murine blastocysts, human cord blood (CB)-derived CD34+ and CD34+ CD38- cells repopulate the hematopoietic tissues of nonimmunocompromised murine embryos and that human donor contribution can persist to adulthood. It is further observed that human hematopoietic progenitor cells are present in murine hematopoietic tissues of midgestational chimeric embryos and that progeny of the injected human HSCs activate erythroid-specific gene expression. Thus, the early murine embryo provides a suitable environment for the survival and differentiation of human CB CD34+ CD38- cells.     

Chronic myelogenous leukemia primitive hematopoietic progenitors demonstrate increased sensitivity to growth factor-induced proliferation and maturation

We investigated whether primary chronic myelogenous leukemia (CML) hematopoietic progenitors demonstrated altered proliferation and maturation in response to growth factor (GF) stimulation.The effect of GF stimulation on proliferation and expansion of committed and primitive progenitors (colony forming cells [CFC]) was evaluated. Culture of CML and normal CD34(+) cells with different GF for 7 days resulted in similar expansion of committed progenitors (CFC). In contrast, GF culture conditions that expanded normal primitive progenitors (week-6 long-term culture-initiating cells (LTC-IC)] led to depletion of CML LTC-IC numbers. GF culture also resulted in increased depletion of week-10 extended LTC-IC, which represent an even more primitive progenitor population, from CML compared with normal CD34(+) cells. CML CD34(+) cells enter into cycle more quickly than normal CD34(+) cells and CML CFC expansion was accelerated compared to normal CFC. Evaluation of primitive progenitor proliferation using PKH-26 and single-cell LTC-IC analysis demonstrated that the majority of CML LTC-IC remaining after GF culture originated from divided CD34(+) cells, whereas GF-cultured normal LTC-IC were derived mainly from undivided cells. Depletion of CML primitive progenitor numbers in association with increased proliferation suggests increased sensitivity to GF-induced maturation.These studies indicate that CML primitive progenitors have enhanced sensitivity to GF-induced cell division and maturation. Altered GF responsiveness may contribute to abnormal expansion of malignant myeloid cells in CML. These findings may also be applied toward the development of novel approaches to select benign stem cells in CML.     

CD7-positive acute myeloid leukemia: further evidence of cellular immaturity

Summary Among 63 cases of acute myeloid leukemia (AML), 14 were found to express the CD7 antigen, a cell surface marker usually found at an early stage during T lineage differentiation. The CD7-positive AML cases consisted of 5 cases of M1, 3 cases of M2, 3 cases of M4, 1 case of M5, 1 case of M6 and 1 case of M7. Among these 63 cases, the proportion of blast cells expressing the CD34 antigen was examined. The proportion of CD34-stained cells among the CD7-positive AML cases, although varying, was significantly larger than that among the CD7-negative AML cases (P<0.05). As the CD34 antigen was expressed on hematopoietic progenitor cells and was considered to reflect an early hematopoietic stage, the high proportion of cells expressing CD34 among the CD7-positive AML cases may support the notion that CD7-positive AML cells are immature.Abbreviations AML acute myeloid leukemia - CD clusters of differentiation Partly supported by grants-in-aid from the Ministry of Education, Culture and Science of Japan (03252102, 63015063, 02256102, and 03670325) and from theFukuoka Anti-Cancer Society     

Interleukin-11 inhibits adipogenesis and stimulates myelopoiesis in human long-term marrow cultures

Interleukin-11 (IL-11) is a bone marrow (BM) stromal-derived growth factor that has been shown to stimulate murine myeloid and lymphoid cells both in vitro and in vivo and to inhibit adipogenesis in a murine fibroblast cell line. We have studied the effects of IL-11 on highly purified human BM stem and progenitor cells and on human long-term marrow cultures (LTMC). Adipocyte differentiation is an integral component of murine and human LTMC. IL-11 stimulates myeloid growth as a single cytokine when added to highly enriched CD34+, HLA-DR+ bone marrow cells. IL-11 stimulated no growth in the more primitive CD34+, HLA-DR- population even in the presence of additional cytokines. IL-11 addition to human LTMC resulted in the expansion of myeloid and mixed, but not erythroid, progenitor populations. IL-11 dramatically increased the adherent cell populations, including both stromal cells and macrophages. Treated cultures also showed marked inhibition of fat accumulation in the adherent cells due in part to a block in the differentiation of preadipocytes to adipocytes, as shown by RNA analysis using adipocyte-specific markers. These data show that IL-11 stimulates a more differentiated, although multipotential, progenitor cell in human BM and that LTMC provide a useful model for studying the effects of this cytokine in the context of the hematopoietic microenvironment.