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.     

KRAS(G12V) enhances proliferation and initiates myelomonocytic differentiation in human stem/progenitor cells via intrinsic and extrinsic pathways

In human hematopoietic malignancies, RAS mutations are frequently observed. Yet, little is known about signal transduction pathways that mediate KRAS-induced phenotypes in human CD34(+) stem/progenitor cells. When cultured on bone marrow stroma, we observed that KRAS(G12V)-transduced cord blood (CB) CD34(+) cells displayed a strong proliferative advantage over control cells, which coincided with increased early cobblestone (CAFC) formation and induction of myelomonocytic differentiation. However, the KRAS(G12V)-induced proliferative advantage was transient. By week three no progenitors remained in KRAS(G12V)-transduced cultures and cells were all terminally differentiated into monocytes/macrophages. In line with these results, LTC-IC frequencies were strongly reduced. Both the ERK and p38 MAPK pathways, but not JNK, were activated by KRAS(G12V) and we observed that proliferation and CAFC formation were mediated via ERK, while differentiation was predominantly mediated via p38. Interestingly, we observed that KRAS(G12V)-induced proliferation and CAFC formation, but not differentiation, were largely mediated via secreted factors, since these phenotypes could be recapitulated by treating non-transduced cells with conditioned medium harvested from KRAS(G12V)-transduced cultures. Multiplex cytokine arrays and genome-wide gene expression profiling were performed to gain further insight into the mechanisms by which oncogenic KRAS(G12V) can contribute to the process of leukemic transformation. Thus, angiopoietin-like 6 (ANGPTL6) was identified as an important factor in the KRAS(G12V) secretome that enhanced proliferation of human CB CD34(+) cells.     

Modelling of ex vivo expansion/maintenance of hematopoietic stem cells

In this study, we described the modelling of the expansion/maintenance of human hematopoietic stem/progenitor cells from adult human bone marrow. CD 34(+)-enriched cell populations from bone marrow were cultured in the presence and absence of human stroma in serum-free media containing bFGF, SCF, LIF and Flt-3 ligand for several days. The cells in the culture were analysed for expansion and phenotype by flow cytometry. Although significant expansion of bone marrow cultures occurred in the presence and absence of human stroma, the results of expansion were effectively better in the presence of a stromal layer. In both situations the phenotypic analysis demonstrated a great expansion of CD 34(+)38(-) cells. The differentiative potential of bone marrow CD 34(+) cells co-cultured with human stroma was primarily shifted towards the myeloid lineage with the presence of CD 15 and CD 33.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin.

The success of stem cell transplantation depends on the ability of i.v. infused stem cells to engraft the bone marrow, a process referred to as homing. Efficient homing requires migration of CD34(+) cells across the bone marrow endothelium, most likely through the intercellular junctions. In this study, we show that loss of vascular endothelial (VE)-cadherin-mediated endothelial cell-cell adhesion increases the permeability of monolayers of human bone marrow endothelial cells (HBMECs) and stimulates the transendothelial migration of CD34(+) cells in response to stromal cell-derived factor-1alpha. Stromal cell-derived factor-1alpha-induced migration was dependent on VCAM-1 and ICAM-1, even in the absence of VE-cadherin function. Cross-linking of ICAM-1 to mimic the leukocyte-endothelium interaction induced actin stress fiber formation but did not induce loss of endothelial integrity, whereas cross-linking of VCAM-1 increased the HBMEC permeability and induced gaps in the monolayer. In addition, VCAM-1-mediated gap formation in HBMEC was accompanied by and dependent on the production of reactive oxygen species. These data suggest that modulation of VE-cadherin function directly affects the efficiency of transendothelial migration of CD34(+) cells and that activation of ICAM-1 and, in particular, VCAM-1 plays an important role in this process through reorganization of the endothelial actin cytoskeleton and by modulating the integrity of the bone marrow endothelium through the production of reactive oxygen species.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Abstract. Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Stem cell maintenance and commitment to differentiation in long-term cultures of murine marrow obtained from different spatial locations in the femur

Murine bone marrow was separated into axial and marginal fractions in order to investigate the ability of cells from different spatial locations in the marrow to establish long-term cultures. The maintenance of haemopoiesis was significantly poor in long-term cultures of marginal marrow compared with axial or control (unfractionated marrow) cultures. Using techniques to further fractionate the axial or marginal marrow by depleting either stromal or haemopoietic cells, it was possible to investigate the relative importance of stromal and haemopoietic cell components. In the combinations studied, the more important determinant of effective in vitro haemopoiesis was the source of the haemopoietic cells rather than the stroma. The most effective stem cell maintenance and commitment to differentiation was observed when the source of the haemopoietic population was axial marrow. The data are consistent with axial marrow being a source of 'high quality' stem cells and this quality being an intrinsic property of the cells rather than one imposed by the stromal environment.     

Contribution of Monocyte/Macrophage Differentiation to the Stromal Layer in Human Long-Term Bone Marrow Cultures

The stroma of bone marrow is a poorly understood tissue which is believed to have important roles in haemopoietic stem cell maintenance and differentiation. We have undertaken immunohistochemical studies of bone marrow stroma in human long-term bone marrow cultures (hLTBMC) and biopsy specimens in order to characterise the cell and matrix components present. We have found two morphological variants of macrophages to be present consistently in hLTBMC stroma, adherent to a substratum of myofibroblastic cells. Large round macrophages appear to actively phagocytic and are formed in hLTBMC regardless of successful establishment of a myofibroblastic cell layer. Elongated macrophages with dendritic processes appear to be non-phagocytic and form only in the presence of a well-established layer of myofibroblasts. Although the functions of these macrophages are not yet known, they have counterparts within intact human bone marrow and their presence in hLTBMC shows some association with the haemopoietic capacity of the cultures.     

Different doses of bone morphogenetic protein 4 promote the expression of early germ cell-specific gene in bone marrow mesenchymal stem cells

Bone morphogenetic protein (BMP)-4 has a crucial role on primordial germ cells (PGCs) development in vivo which can promote stem cell differentiation to PG-like cells. In this study, we investigated the expression of Mvh as one of the specific genes in primordial germ cells after treatment with different doses of BMP4 on bone mesenchymal stem cells (BMSCs)-derived PGCs. Following isolation of BMSCs from male mouse femur and tibia, cells were cultured in medium for 72 h. Passage 4 murine BMSCs were characterized by CD90, CD105, CD34, and CD45 markers and osteo-adipogenic differentiation. Different doses of BMP4 (0, 0.01, 0.1, 1, 5, 25, 50, and 100 ng/ml) were added to BMSCs for PGCs differentiation during 4-days culture. Viability percent, proliferation rates, and expression of Mvh gene were analyzed by RT-qPCR. Data analysis was done with ANOVA test. CD90⁺, CD105⁺, CD34⁻, and CD45⁻ BMSCs were able to differentiate to osteo-adipogenic lineages. The results revealed that proliferation rate and viability percent were raised significantly (p ≤ 0.05) by adding 1, 5, 25 ng/ml of BMP4 and there were decreased to the lowest rate after adding 100 ng/ml BMP4 (p ≤ 0.05). There were significant up regulation (p ≤ 0.05) in Mvh expression between 25, 50, and 100 ng/ml BMP4 with other doses. So the selective dose of BMP-4 for treatment during 4-day culture was 25 ng/ml. The results suggest that addition of 25 ng/ml BMP4 had the best effects based on gene-specific marker expression.     

Heterogeneity of stromal precursor cells isolated from rat bone marrow

Bone marrow stroma contains mesenchymal stem cells (MSC) which are precursor for at least mesenchyma-derived cells. Recent investigations revealed a lot of questions concerning MSC biology that should be further refined. The aim of this study was the comparative analysis of rat bone marrow stroma cells cultures. Mesenchymal precursor cells isolated from rat bone marrow were passed up to 50 times. Comparative morphological and immunophenotypical analysis of these cultures was carried out as well as their ability to osteogenic differentiation was studied. The isolated cultures contained morphologically different types of cells and thus showed a high heterogenity level. Morphology of these cell types was described. The heterogeneity level was reported to decrease over time. It was found out that subcultures isolated from different rats shared the same immunophenotype characteristics (CD90+, CD44+, CD54+, CD 106+, CD45-, CD11b-), but differed in their morphology as well as in ability to osteogenic differentiation. Thus MSC identification requires more specific marker and functional tests to be used.     

Expression of the cell adhesion molecule E-cadherin by the human bone marrow stromal cells and its probable role in CD34(+) stem cell adhesion.

Bone marrow stroma is the physical basis of the haematopoietic microenvironment and regulates several key features of stem cell proliferation and differentiation. It plays a crucial role in maintaining haematopoietic homeostasis. Earlier studies have shown that this is achieved through interactions with the extracellular matrix and specific molecules called the cell adhesion molecules (CAMs). In this paper, we show that E-cadherin, a cell adhesion molecule which plays a crucial role in cell-cell aggregation during development, is also present in the bone marrow stroma. The expression of the CAM can also be demonstrated on a subset of CD34(+)stem cells. Stromal expression of E-cadherin is decreased when treated with lymphokine mixture, phytohaemagglutinin-treated-leukocyte-conditioned medium (PHA-LCM). This is the reverse of ICAM-I expression, which increases with PHA-LCM treatment. E-cadherin shows homotypic and homophilic interaction and its presence on a subset of CD34(+)cells leads to speculation on whether this CAM has a role in adherence of primitive stem cells to the marrow stroma.     

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.     

Increased reactivity of cultured chicken blastodermal cells to anti-stage-specific embryonic antigen-1 antibody after exposure to bone morphogenetic proteins

We evaluated whether bone morphogenetic proteins (BMPs) increased the reactivity of chicken stage X blastodermal cells to the germ cell marker, anti-stage-specific embryonic antigen (SSEA)-1 antibody. In Experiment 1, blastodermal cells cultured on a feeder layer of SIM mouse embryo-derived thioguanine and ouabain resistant (STO) cells were treated with different doses of BMP-2 and/or BMP-4, and the anti-SSEA-1 antibody reactivity of cultured cells was examined 48 h later. A significant (P < 0.05) increase in the number of anti-SSEA-1 antibody-positive cells was detected after the addition of 75 or 100 ng/ml BMP-2. Neither 0-20 ng/ml BMP-4 nor the combined addition of 75 ng/ml BMP-2 with either 10 or 15 ng/ml BMP-4 increased reactivity more than that induced by 75 ng/ml BMP-2 alone. Results of the qualification and quantification of BMP receptor kinase (BRK)-1, BRK-2, and BRK-3 using RT-PCR and real-time PCR showed that all three receptors were detected in blastodermal cells treated with BMPs, intact stage X embryos and 5.5-day-old embryonic gonads, but no expression was detected in STO feeder cells. In Experiment 2, the treatment of stage X embryos with different doses of BMP-2 (0.15-3 ng/embryo) or BMP-4 (0.02-0.4 ng/embryo) did not affect the reactivity of 5.5-day-old embryonic gonadal cells to the anti-SSEA-1 antibody. BRK-1 expression was selectively increased in stage X embryos after the infusion of 3ng BMP-2 than after no infusion, but no changes in other BRKs' expression were detected. In conclusion, the addition of BMP-2 to culture medium in the presence of STO feeder cells promoted the reactivity of blastodermal cells to anti-SSEA-1 antibody, which might contribute to the generation of chicken primordial germ cell precursor or germ cell-like cells. The relationship between BMP action and BRK expression was further discussed.     

Vascular cell adhesion molecule-1 expression and hematopoietic supportive capacity of immortalized murine stromal cell lines derived from fetal liver and adult bone marrow

Ontogeny-specific differences in hematopoietic behavior may be influenced by unique adhesive interactions between hematopoietic cells and the microenvironment, such as that mediated by vascular cell adhesion molecule-1 (VCAM-1, CD 106). Although VCAM-1 is variably expressed during vertebrate development, we hypothesized that VCAM-1 expression might be linked to the enhanced capacity of the fetal liver microenvironment to support hematopoiesis. To test this we used immortalized murine stromal cell lines derived from midgestation fetal liver and adult bone marrow to compare the functional expression of VCAM-1. Molecular analysis of VCAM-1 expression was performed on stromal cell lines using Northern blot analysis, immunoprecipitation studies, and solid-phase enzyme-linked immunosorbent assay. Hematopoietic studies were performed by coculturing fetal liver cells with stromal cell lines, and the functional readout was determined by high-proliferative potential colony-forming cell (HPP-CFC) adherence assays. In contrast to our initial hypothesis, we observed greater expression of VCAM-1 messenger ribonucleic acid and protein on an adult marrow stromal cell line. In functional studies, anti-VCAM-1 antibody inhibited the binding of nearly half of the HPP-CFCs to adult marrow stroma but had a minimal effect on their binding to fetal liver stroma, despite the greater adherence of HPP-CFCs to fetal stroma. We conclude that VCAM-1 influences the hematopoietic supportive capacity of immortalized murine stroma derived from adult bone marrow. Our studies suggest that cellular interactions other than those mediated by VCAM-1 are involved in the increased adhesive capacity of immortalized murine stroma derived from fetal liver.     

Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment

The content of stem cells was analysed in bone marrow samples from 75 multiple myeloma patients. In unstimulated bone marrow the percentage of CD34+cells was significantly reduced in 11 patients previously treated with melphalan-prednisolone (MP)(median= 0.15%) compared to median 0.87% in 31 untreated patients (P=0.0001). The bone marrow cellularity in the two groups did not differ. There was no correlation between the number of courses or total dose of melphalan and content of CD34+cells in the bone marrow. The clonogenicity as, well as the ability to expand the marrow stem cell pool during growth factor treatment were also reduced in MP treated patients compared to untreated patients. Analysis of different subsets of CD34+ cells revealed no influence on the pre B cell compartment in the bone marrow by MP treatment, but the committed stem cells (CD34+CD38+) were reduced more than the uncommitted stem cells (CD34+CD38—) in the MP treated group compared to the untreated patients. Mobilisation to and harvest of total number of CD34+ cells from peripheral blood was also reduced in the MP treated group. There was, however, no difference in the distribution between CD34+CD38+and CD34+CD38—populations in the leukapheresis products in the untreated and the melphalan-treated group, suggesting selective mobilisation of CD34+CD38+ cells and/or differentiation of CD34+ CD38-cells during growth factor stimulation. We conclude that melphalan decreased the number of stem cells in the bone marrow, the ability to expand the stem cell pool and mobilise stem cells to the pheripheral blood.     

Bone morphogenetic protein (BMP)-7 but not BMP-2 and BMP-4 improves maintenance of primitive peripheral blood-derived hematopoietic progenitor cells (HPC) cultured in serum-free medium supplemented with early acting cytokines

BMPs regulate the developmental program of hematopoiesis. We demonstrate an increased expression of the BMP receptors Ia and II on cultured CD34⁺ cells and examine the impact of BMP-2, -4 and -7 on postnatal HPC cultured with stem cell factor, flt3-ligand and interleukin-3 (SF3). The addition of BMP-2 at 5, 25 and 50 ng/m to serum-free medium with SF3 yielded a 1.4- to 1.2-fold increase of CD34⁺ cells after seven days, but no effect on CFC or LTC-IC was observed. BMP-4 at 25 ng/ml induced a 2.9-fold expansion of colony-forming cells (CFC) within 1 week followed by a decrease to pre-culture values on day 14. The number of long-term culture initiating cells (LTC-IC) decreased by the factor 40 from day 0 to day 14. BMP-7 at 5–50 ng/ml had not effect on the expansion of CD34⁺ cells and CFC, but improved at 5 ng/ml the survival of LTC-IC significantly as compared to SF3 alone. In summary, BMP-2, -4 and -7 have no effect on the proliferation of CD34⁺ cells and CFC cultured with serum-free medium and SF3. However, BMP-7 but not BMP-2 and BMP-4 prevents the loss of primitive hematopoietic progenitor cells cultured in SFM plus SF3.     

Human bone marrow osteoprogenitors express estrogen receptor-alpha and bone morphogenetic proteins 2 and 4 mRNA during osteoblastic differentiation.

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. This study examines some aspects of the differentiation in vitro of human bone marrow fibroblastic cells cultured in the absence (basal media) or presence of 1nM dexamethasone and 50 micrograms/ml ascorbate for 6, 10, 14, and 21 days. Northern blot analysis and in situ hybridisation with digoxygenin-labelled riboprobes for Type I collagen, osteocalcin, bone morphogenetic proteins 2 (BMP-2), and 4 (BMP-4) and the estrogen receptor alpha (ERalpha), together with immunocytochemical analysis of ERalpha expression and histochemical staining of alkaline phosphatase was performed. In basal media, alkaline phosphatase activity and collagen expressions were detected at day 6, ERalpha from day 10 and osteocalcin from day 10. In the presence of dexamethasone and ascorbate, cell proliferation and alkaline phosphatase were markedly stimulated over 10 to 14 days with a dramatic increase in the temporal expression of Type I collagen, ERalpha, and osteocalcin mRNAs in these cultures. Northern blot analysis showed cells cultured in basal media, expressed the highest levels of the mRNA for each marker protein at day 14, whereas in the presence of ascorbate and dexamethasone, the highest levels for alkaline phosphatase, ERalpha, osteocalcin, BMP-2, and BMP-4 were observed at day 21. ERalpha, BMP-2, and BMP-4 expression were found to correlate temporally with induction of the osteoblast phenotype as determined by alkaline phosphatase, collagen, and osteocalcin expression. These results give additional information on the development of the osteoblast phenotype from early fibroblastic stem cells and on the biological factors involved in this process. These studies suggest a role for estrogen and BMP-2 and -4 in the differentiation of osteoprogenitor cells.     

Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells.

It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.