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.     

Xyloside effects on in vitro hematopoiesis: functional and biochemical studies

Xyloside supplementation of long-term bone marrow cultures (LTBMCs) has been reported to result in greatly enhanced proliferation of hematopoietic stem cells. This was presumed to be the result of xyloside-mediated perturbation of proteoglycan synthesis by marrow-derived stromal cells. To investigate this phenomenon, we first studied the effects of xyloside supplementation on proteoglycan synthesis by D2XRadII bone marrow stromal cells, which support hematopoietic stem cell proliferation in vitro. D2XRadII cells were precursor labelled with 35S-sulfate, and proteoglycans separated by ion exchange chromatography, isopyknic CsCl gradient centrifugation, and gel filtration HPLC. Xyloside-supplemented cultures showed an approximately fourfold increase in total 35S incorporation, mainly as free chondroitin-dermatan sulfate (CS/DS) glycosaminoglycan chains in the culture media. Both xyloside supplemented and nonsupplemented cultures synthesized DS1, DS2, and DS3 CS/DS proteoglycans as previously described. In contrast to previous reports, xyloside was found to inhibit hematopoietic cell growth in LTBMC. Inhibitory effects were observed both in cocultures of IL-3-dependent hematopoietic cell lines with supportive stromal cell lines and in primary murine LTBMCs. Xyloside was found to have a marked inhibitory effect on the growth of murine hematopoietic stem cells and IL-3-dependent hematopoietic cell lines in clonal assay systems and in suspension cultures. In contrast, dialyzed concentrated conditioned media from LTBMCs had no such inhibitory effects. These findings suggest that xyloside-mediated inhibition of hematopoietic cell growth in LTBMC resulted from a direct effect of xyloside on proteoglycan synthesis by hematopoietic cells.     

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1α induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1α induced IL-1α and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34⁺ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment. J. Cell. Physiol. 176:57–66, 1998. © 1998 Wiley-Liss, Inc.     

Human marrow stromal precursors are alpha 1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.     

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice.

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Persistent production of colony-stimulating factor (CSF-1) by cloned bone marrow stromal cell line D2XRII after X-irradiation

The adherent stromal layer in long-term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony-stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 X 10(6) D2XRII cells in 8.0 ml produced CSF-1 (M-CSF) at around 100-150 units/0.1 ml medium. Following X-irradiation there was a dose-dependent decrease in the production of CSF-1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X-ray dose reducing CSF-1 production to 50% was 100-fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X-irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF-1. The data indicate significant divergence of two biologic effects of X-irradiation on plateau-phase marrow stromal cells: physiologic function of adherence and CSF-1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X-irradiation-associated marrow suppression and leukemogenesis.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Surface protein expression between human adipose tissue-derived stromal cells and mature adipocytes

Adipose tissue contains a stroma that can be easily isolated. Thus, human adipose tissue presents an source of multipotent stromal cells. In order to determine the implication of hematopoietic markers in adipocyte biology, we have defined part of the phenotype of the human adipose tissue-derived stromal cells, and compared this to fully differentiated adipocytes. Flow cytometry demonstrates that the protein expression phenotype of both cell types are similar and includes the expression of CD10, CD13, CD34, CD36, CD55, CD59 and CD65. No significant difference between subcutaneous and omental adipose tissue could be demonstrated concerning the expression of these markers. However, the expression of CD34, CD36 and CD65 is cell-dependent. While the expression of CD36 and CD65 doubled between stromal cells and mature adipocytes, the expression of CD34 decreased, despite this protein being present on the mature adipocyte. As CD34 is described as a stem cell marker and it being unlikely to be expressed on differentiated cells, this result was confirmed by immunostaining and western blot. The clear function of this protein on the adipocyte membrane remains to be determined. The characterization of new proteins on mature adipocytes could have broad implications for the comprehension of the biology of this tissue.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

Bone marrow mesenchymal stem cells for improving hematopoietic function: an in vitro and in vivo model. Part 2: Effect on bone marrow microenvironment

The aim of the present study was to determine how mesenchymal stem cells (MSC) could improve bone marrow (BM) stroma function after damage, both in vitro and in vivo. Human MSC from 20 healthy donors were isolated and expanded. Mobilized selected CD34(+) progenitor cells were obtained from 20 HSCT donors. For in vitro study, long-term bone marrow cultures (LTBMC) were performed using a etoposide damaged stromal model to test MSC effect in stromal confluence, capability of MSC to lodge in stromal layer as well as some molecules (SDF1, osteopontin,) involved in hematopoietic niche maintenance were analyzed. For the in vivo model, 64 NOD/SCID recipients were transplanted with CD34+ cells administered either by intravenous (i.v.) or intrabone (i.b.) route, with or without BM derived MSC. MSC lodgement within the BM niche was assessed by FISH analysis and the expression of SDF1 and osteopontin by immunohistochemistry. In vivo study showed that when the stromal damage was severe, TP-MSC could lodge in the etoposide-treated BM stroma, as shown by FISH analysis. Osteopontin and SDF1 were differently expressed in damaged stroma and their expression restored after TP-MSC addition. Human in vivo MSC lodgement was observed within BM niche by FISH, but MSC only were detected and not in the contralateral femurs. Human MSC were located around blood vessels in the subendoestal region of femurs and expressed SDF1 and osteopontin. In summary, our data show that MSC can restore BM stromal function and also engraft when a higher stromal damage was done. Interestingly, MSC were detected locally where they were administered but not in the contralateral femur.     

Hyaluronan expressed by the hematopoietic microenvironment is required for bone marrow hematopoiesis

The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2(flox/flox);Has1(-/-);Has3(-/-) triple knock-out (tKO) mice as compared with wild type (WT) and Has1(-/-);Has3(-/-) double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment.     

Multipotent marrow stromal cell line is able to induce hematopoiesis in vivo.

Several murine marrow stromal cells were established from murine bone marrow cultures. Stromal cell lines transfected with a tumor-inducing polyoma virus middle T antigen (MTAg) were inoculated into nude mice subcutaneously. KUSA-MTAg cells, one of these cell lines, led to the rapid local development of bone marrow consisting of trilineage hematopoietic cells and bone; other cell lines produced spindle cell sarcoma or hemangiosarcoma. These results suggested that a single stromal cell line, KUSA-MTAg cells, may induce hematopoietic stem cells or early progenitors of three lineages of hematopoietic cells in vivo. Interestingly, untransfected KUSA cells expressed three new mesenchymal phenotypes, osteocytes, adipocytes, and myotubes, after treatment with 5-azacytidine.     

CD34+ fibrocytes: morphology, histogenesis and function

The connective tissue of virtually all human organs harbors huge amounts of resident CD34(+) fibrocytes. Recent studies have shown that CD34(+) fibrocytes derive from circulating CD14(+) monocytes. CD34(+) fibrocytes are involved in wound healing, act as antigen presenting cells and secrete a multitude of cytokines. Due to their diverse functions CD34(+) fibrocytes play a role in connective tissue diseases, pulmonary fibrosis and tumor associated stromal remodeling. Stromal remodeling precipitated by invasive carcinomas is characterized by a loss of CD34(+) expression paralleled by a gain of alpha-SMA expression in stromal cells resulting in a phenotype change from CD34(+) fibrocytes towards alpha-SMA positive myofibroblasts. This process is very stereotypic and may play an essential role in local tumor invasion and systemic dissemination, since a reduction of antigen presenting CD34(+) fibrocytes might constitute a step in escaping the hosts' immune control directed against invasive carcinoma cells.     

In vitro differentiation of mesenchymal progenitor cells derived from porcine umbilical cord blood

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.