Similarities in long-term cultures of blood and bone marrow from patients with acute myelogenous leukemia

Dexter-type long-term cultures (LTC) were initiated with peripheral blood (PB) and/or bone marrow cells from 11 patients with acute myelogenous leukemia (AML), and 2 with myelodysplastic syndrome in blastic transformation. Marrow and PB cells from normal subjects served as controls. Assessment of nucleated cells and clonogenic progenitors in the adherent and nonadherent fractions of LTC revealed active hemopoiesis for greater than 5 wks in 4 of 8 cultures of AML blood, and 4 of 7 of AML marrow. The morphology and kinetics of nucleated cells and progenitors with putative normal (granulocyte-macrophage colony-forming units or CFU-gm), and abnormal (blast) phenotype in LTC from AML blood were similar to those from AML marrow, and adherent cells positive for collagen I and III and vimentin were found in both types of LTC. Growth of CFU-gm colonies ceased by wk 5-8 in AML cultures, significantly earlier than in LTC of normal marrow cells (survival of greater than 10 wks), which may indicate derivation of the CFU-gm from a transformed clone or deficiency of stromal function in the leukemic state. In most AML blood and AML marrow LTCs, growth of abnormal (blast) colonies continued until wk 4-6. This study demonstrates certain similarities of morphology and function between LTC of AML blood and AML marrow cells. LTC may provide a useful model for further analysis of circulating primitive hemopoietic progenitor cells in leukemic states.     

Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.     

Substrate elasticity provides mechanical signals for the expansion of hemopoietic stem and progenitor cells

Surprisingly little is known about the effects of the physical microenvironment on hemopoietic stem and progenitor cells. To explore the physical effects of matrix elasticity on well-characterized primitive hemopoietic cells, we made use of a uniquely elastic biomaterial, tropoelastin. Culturing mouse or human hemopoietic cells on a tropoelastin substrate led to a two- to threefold expansion of undifferentiated cells, including progenitors and mouse stem cells. Treatment with cytokines in the presence of tropoelastin had an additive effect on this expansion. These biological effects required substrate elasticity, as neither truncated nor cross-linked tropoelastin reproduced the phenomenon, and inhibition of mechanotransduction abrogated the effects. Our data suggest that substrate elasticity and tensegrity are important mechanisms influencing hemopoietic stem and progenitor cell subsets and could be exploited to facilitate cell culture.     

Bone marrow clonogenic capability, cytokine production, and thymic output in patients with common variable immunodeficiency

In patients with primary Ab deficiencies, hematological and immunological abnormalities are frequently observed. A regenerative failure of hemopoietic stem/progenitor cells has been hypothesized. We evaluated in the bone marrow (BM) of 11 patients with common variable immunodeficiency, the phenotype of BM progenitors and their in vitro growth by colony-forming cell (CFC) and long-term culture (LTC) assays. A significant decrease in erythroid and mixed CFC and, to a greater extent, in primitive LTC-CFC progenitors was observed in patients compared with healthy controls. The frequency of BM pre-B and pro-B cells correlated directly with the absolute number of CD19+ lymphocytes. BM cells cultured in vitro produced spontaneously lower amounts of IL-2 and elevated levels of TNF-alpha compared with controls, indicating a skewing toward a proapoptotic cytokine pattern. In addition, stromal cells generated after BM LTC secreted less IL-7 and displayed by immunohistochemistry an altered phenotype. These findings were associated with a significant decrease in naive Th cells coexpressing CD31 in the peripheral blood. These results indicate an impaired growth and differentiation capacity of progenitor cells in patients with common variable immunodeficiency.     

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.     

Characterization of thymic progenitors in adult mouse bone marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1(-)) (Thy1.1(-) progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1(-) progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1(-) population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.     

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Comparison of direct and indirect radiation effects on osteoclast formation from progenitor cells derived from different hemopoietic sources

Hemopoietic stem and progenitor cells from different sources differ in radiosensitivity. Recently, we have demonstrated that the multinucleated cell responsible for bone resorption and marrow cavity formation, the osteoclast, is in fact of hemopoietic lineage. In this investigation we have studied the radiosensitivity of osteoclast formation from two different hemopoietic tissues: fetal liver and adult bone marrow. Development of osteoclasts from hemopoietic progenitors was induced by coculture of hemopoietic cell populations with fetal mouse long bones depleted of their own osteoclast precursor pool. During culture, osteoclasts developed from the exogenous cell population and invaded the calcified hypertrophic cartilage of the long bone model, thereby giving rise to the formation of a primitive marrow cavity. To analyze the radiosensitivity of osteoclast formation, either the hemopoietic cells or the bone rudiments were irradiated before coculture. Fetal liver cells were found to be less radiosensitive than bone marrow cells. The D0, Dq values and extrapolation numbers were 1.69 Gy, 5.30 Gy, and 24.40 for fetal liver cells and 1.01 Gy, 1.85 Gy, and 6.02 for bone marrow cells. Irradiation of the (pre)osteoclast-free long bone rudiments instead of the hemopoietic sources resulted in a significant inhibition of osteoclast formation at doses of 4 Gy or more. This indirect effect appeared to be more prominent in the cocultures with fetal than with adult hemopoietic cells. Furthermore, radiation doses of 8.0-10.0 Gy indirectly affected the appearance of other cell types (e.g., granulocytes) in the newly formed but underdeveloped marrow cavity. The results indicate that osteoclast progenitors from different hemopoietic sources exhibit a distinct sensitivity to ionizing irradiation. Radiation injury to long bone rudiments disturbs the osteoclast-forming capacity as well as the hemopoietic microenvironment.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures

B lymphocytes are not produced in the Dexter long-term bone marrow cultures, but a primitive B cell precursor is present. The findings presented in this study demonstrate that this precursor can be induced to produce B lymphocytes by transferring the cultures to the Whitlock conditions for the long-term growth of B cells in vitro. Two weeks after the transfer of cultures maintained at 33 degrees C in medium supplemented with horse serum and steroids to low concentrations of fetal calf serum at 37 degrees C, marked effects can be observed. The pattern of cell growth changes from one in which the hemopoietic cells are clustered in tight foci containing several hundred cells to smaller ones in which the cells are not as densely packed. Fat cells in the adherent layer disappear and the supporting stroma becomes more uniform in appearance. This change in the culture format is accompanied by a decrease in the number of nonadherent cells and a shift from myelopoiesis to lymphopoiesis. The numbers of granulocyte-macrophage progenitors decline weekly after the change in culture conditions and are not detected by the third week. B cell colony-forming units appear by 3 wk. Cells that express the 14.8 cell surface antigen are induced by 1 wk after the change in culture conditions, followed by the appearance of surface IgM-bearing cells 2 wk later. This shift to lymphopoiesis can be confirmed morphologically. Granulocytes and macrophages disappear from the cultures by 4 wk, at which time almost all of the cells have a characteristic lymphocyte morphology. Upon switching these cultures back to the original Dexter conditions, only low levels of transient myelopoiesis can be reinitiated.     

An in vitro assay for T lymphocyte progenitors (CFU-preT)

Thy-1.2 negative progenitors give rise to Thy-1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen-stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy-1 positive, peanut agglutinin-positive, and active in a pre-T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU-preT.     

Hemopoietic environment necessary for an early stage in differentiation of antibody-forming cells: effect of cortisol

Treatment of adult mice with cortisol decreased the number and frequency of bone marrow precursors of anti-sheep erythrocyte hemolytic plaque-forming cells (P-PFC) and marrow “lymphocytes.” However, the more primitive progenitors of P-PFC in marrow were not affected. Treatment of prospective recipients of marrow grafts with cortisol impaired the generation of P-PFC by transplanted primitive progenitor cells. Differentiation with proliferation of P-PFC into mature PFC was less dramatically affected by cortisol. P-PFC were not apparently suppressed directly, since the fall-off of P-PFC did not occur during the first day after administration of cortisol. Thus, cortisol impairs the generation of P-PFC by altering a hemopoietic inductive environment necessary for this step in differentiation of immunocompetent cells.     

Characterization of the in vitro stromal microenvironment of human bone marrow

Utilizing long-term in vitro culture techniques, we characterized the cellular composition and functional attributes of the human in vitro bone marrow stromal microenvironment. Morphologic, specific cytochemical and immunologic methods demonstrated that the marrow stromal adherent layer (AL) reached confluency at two to three weeks, and was comprised of 60%-70% fibroblastic cells, 10%-20% endothelial cells, 10%-20% monocyte/macrophages and 5%-10% fat-laden adherent cells. These proportions of cell types persisted for at least three months concomitant with proliferation of CFU-gm and BFU-e. In contrast, umbilical cord blood cells did not form a stromal AL despite persistence of hemopoietic progenitor cell proliferation. These findings provide a basis for improved understanding of cellular interactions regulating hemopoiesis.     

Why clinicians should be interested in interleukin-3

Interleukin-3 (IL-3), a product of activated immune cells has recently been cloned and introduced in preclinical and clinical trials. The biological target-cell spectrum of IL-3 is broad and includes progenitor cells of various hematopoietic lineages as well as multiple stages of stem cell differentiation. IL-3 also induces growth of most primitive hemopoietic progenitors (CFU-blast). Synergistic effects on growth of myeloid cells (i.e. macrophages, eosinophils and blood basophils) are obtained by sequential use of IL-3 and later-acting myelopoietic cytokines. In addition, IL-3 supports terminal maturation, prolongs survival and enhances the functional properties of myeloid cells through high-affinity binding sites. In vivo administration of IL-3 is followed by an increase in peripheral white blood cell counts as well as by an increase in the number of circulating progenitor cells giving rise to mature hemopoietic cells in response to more lineage-restricted growth factors. IL-3 also regulates growth of leukemic cells and primes them to become more sensitive to cell cycle specific cytotoxic drugs. IL-3 apparently represents a novel and unique hemopoietic growth factor. Its clinical use should offer new strategies in the treatment of cytopenia, leukemic disease and in stem cell transplantation.     

Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

Transgenic expression of stromal cell-derived factor-1/CXC chemokine ligand 12 enhances myeloid progenitor cell survival/antiapoptosis in vitro in response to growth factor withdrawal and enhances myelopoiesis in vivo

Hemopoiesis is regulated in part by survival/apoptosis of hemopoietic stem/progenitor cells. Exogenously added stromal cell-derived factor-1 ((SDF-1)/CXC chemokine ligand (CXCL)12) enhances survival/antiapoptosis of myeloid progenitor cells in vitro. To further evaluate SDF-1/CXCL12 effects on progenitor cell survival, transgenic mice endogenously expressing SDF-1/CXCL12 under a Rous sarcoma virus promoter were produced. Myeloid progenitors (CFU-granulocyte-macrophage, burst-forming unit-erythroid, CFU-granulocyte-erythrocyte-megakaryocyte-monocyte) from transgenic mice were studied for in vitro survival in the context of delayed addition of growth factors. SDF-1-expressing transgenic myeloid progenitors were enhanced in survival and antiapoptosis compared with their wild-type littermate counterparts. Survival-enhancing effects were due to release of low levels of SDF-1/CXCL12 and mediated through CXCR4 and G(alpha)i proteins as determined by ELISA, an antagonist to CXCR4, Abs to CXCR4 and SDF-1, and pertussis toxin. Transgenic effects of low SDF-1/CXCR4 may be due to synergy of SDF-1/CXCL12 with other cytokines; low SDF-1/CXCL12 synergizes with low concentrations of other cytokines to enhance survival of normal mouse myeloid progenitors. Consistent with in vitro results, progenitors from SDF-1/CXCL12 transgenic mice displayed enhanced marrow and splenic myelopoiesis: greatly increased progenitor cell cycling and significant increases in progenitor cell numbers. These results substantiate survival effects of SDF-1/CXCL12, now extended to progenitors engineered to endogenously produce low levels of this cytokine, and demonstrate activity in vivo for SDF-1/CXCL12 in addition to cell trafficking.     

Cutting edge: activation of the p38 mitogen-activated protein kinase signaling pathway mediates cytokine-induced hemopoietic suppression in aplastic anemia

Myelosuppressive cytokines, in particular IFN-gamma and TNF-alpha, play an important role in the pathogenesis of idiopathic aplastic anemia in humans. It is unknown whether these negative regulators of hemopoiesis suppress stem cells by activating a common signaling cascade or via distinct nonoverlapping pathways. In this study, we provide evidence that a common element in signaling for IFN-gamma and TNF-alpha in human hemopoietic progenitors is the p38/MapKapK-2 signaling cascade. Our studies indicate that pharmacological inhibition of p38 reverses the suppressive effects of IFN-gamma and TNF-alpha on normal human bone marrow-derived erythroid and myeloid progenitors. Most importantly, inhibition of p38 strongly enhances hemopoietic progenitor colony formation from aplastic anemia bone marrows in vitro. Thus, p38 appears to play a critical role in the pathogenesis of aplastic anemia, suggesting that selective pharmacological inhibitors of this kinase may prove useful in the treatment of aplastic anemia and other cytokine-mediated bone marrow failure syndromes.