Proteoglycan synthesis in two murine bone marrow stromal cell lines

There is evidence indicating that stromal proteoglycans are an important functional component of the hematopoietic microenvironment. Proteoglycan synthesis was therefore investigated in the MS3-2A and D2XRII hematopoietic stromal cell lines. These lines differ in their capacity to support hematopoiesis in vitro, D2XRII supporting in vitro hematopoiesis, whereas MS3-2A does not. Cells were labeled with 35S- sulfate as precursor, and 4 mol/L guanidine HCl extracts of cells and media were analyzed by ion-exchange chromatography, cesium chloride density gradient centrifugation, and molecular sieve chromatography. Proteoglycans were further examined by enzymatic and chemical digestions. MS3-2A cells produced at least three proteoglycan species. Two chondroitin/dermatan sulfate (CS/DS) proteoglycans, Kav = 0.40 and Kav = 0.68 on Sepharose CL-2B, were present primarily in the medium. The respective glycosaminoglycan molecular weight (mol wt) values were 38 kd and 40 kd. A heparan sulfate (HS) proteoglycan of Kav = 0.58 and glycosaminoglycan mol wt 36 kd was present primarily in the cell layer extract. D2XRII cells synthesized two HS proteoglycans. The larger (Kav = 0.45; glycosaminoglycan mol wt, 30 kd) was of low density on gradient centrifugation and more prominent in the cell layer extracts, whereas the smaller (Kav = 0.68; glycosaminoglycan mol wt, 38 kd) was dense and present mainly in the culture medium. A single CS/DS proteoglycan species of Kav 0.78 and average glycosaminoglycan of mol wt 18 kd was present in roughly equal amounts in the medium and in the cell layer. MS3-2A and D2XRII thus appear phenotypically distinct with respect to proteoglycan synthesis. These differences are discussed in relation to the microenvironmental function of bone marrow stromal elements.     

A novel 37-Kd adhesive membrane protein from cloned murine bone marrow stromal cells and cloned murine hematopoietic progenitor cells

The adhesion of hematopoietic progenitor cells to bone marrow stromal cells is critical to hematopoiesis and involves multiple effector molecules. Stromal cell molecules that participate in this interaction were sought by analyzing the detergent-soluble membrane proteins of GBI/6 stromal cells that could be adsorbed by intact FDCP-1 progenitor cells. A single-chain protein from GBI/6 cells having an apparent molecular weight of 37 Kd was selectively adsorbed by FDCP-1 cells. This protein, designated p37, could be surface-radiolabeled and thus appeared to be exposed on the cell membrane. An apparently identical 37- Kd protein was expressed by three stromal cell lines, by Swiss 3T3 fibroblastic cells, and by FDCP-1 and FDCP-2 progenitor cells. p37 was selectively adsorbed from membrane lysates by a variety of murine hematopoietic cells, including erythrocytes, but not by human erythrocytes. Binding of p37 to cells was calcium-dependent, and was not affected by inhibitors of the hematopoietic homing receptor or the cell-binding or heparin-binding functions of fibronectin. It is proposed that p37 may be a novel adhesive molecule expressed on the surface of a variety of hematopoietic cells that could participate in both homotypic and heterotypic interactions of stromal and progenitor cells.     

Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.

Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.     

Biosynthesis of proteoglycans by rat granuloma cells cultured in vitro: modulation by gonadotropins, steroid hormones, prostaglandins, and a cyclic nucleotide

Rat ovarian granulosa cells were isolated from immature female rats 48 h after stimulation with 5 IU PMS gonadotropin and then maintained in culture. The effects of ovine (o)FSH, oLH, hCG, testosterone, 17 beta-estradiol, progesterone, prostaglandins E1, E2, F1 alpha, F2 alpha, N,O'-dibutyryl cAMP, and theophylline on proteoglycan synthesis by the granulosa cells in vitro were examined using [35S]sulfate as a precursor. oFSH, oLH, hCG, prostaglandins E1 and E2, N,O'-dibutyryl cAMP, theophylline and testosterone stimulated the production of 35S-labeled proteoglycans compared to control cultured. 17 beta-Estradiol, progesterone, and the prostaglandins F1 alpha, and F2 alpha, showed no stimulation. Two major species of proteoglycan are synthesized and secreted into the medium: 1) a population with large hydrodynamic size (Kav = 0.30 on Sepharose CL-2B); and 2) a population with relatively small size (Kav = 0.60 on Sepharose CL-2B). The stimulatory effect of the hormones was accounted for entirely by a net increase in synthesis of the smaller proteoglycan population. The hormones exert their stimulatory effects on proteoglycan synthesis at concentrations similar to those required for other in vitro biological responses of granulosa cells.     

Effect of the irradiated microenvironment on the expression and retrotransposition of intracisternal type A particles in hematopoietic cells

We have previously demonstrated that the frequency of transformation of the factor-dependent hematopoietic cell line FDCP-1JL26 was dramatically increased when cells were cocultured with the irradiated bone marrow cell line D2XRII. In many of our factor-independent subclonal cell lines that we examined, transformation to factor independence appeared to be due to the retrotransposition of intracisternal type A particles (IAP) into the growth factor genes that are normally required for survival and growth of FDCP-1JL26 cells. To determine the role of the irradiated microenvironment in the evolution of factor-independent cells, we have examined the expression and retrotransposition of IAPs after exposure to the irradiated bone marrow stromal cell line D2XRII. Differential display and Northern blot analysis demonstrated that IAPs were overexpressed in a nonautocrine factor-independent subclonal cell line, FI7CL2. The frequency of retrotransposition was determined by the introduction of the IAP-neo(RT) plasmid into FDCP-1JL26 cells. The IAP-neo(RT) contains a neomycin resistance gene (neo) that only becomes active after retrotransposition, and thus the frequency of retrotransposition in FDCP-1JL26 cells was quantified by determining the frequency of neo-resistant cells.No significant increases in the expression of IAPs were observed after the cells were exposed to the irradiated stromal cells. This observation is in agreement with the observation that no increase in the frequency of retrotransposition could be detected. These results suggest that the irradiated bone marrow may have a passive role in the selection of factor-independent cells. During cocultivation, bone marrow stromal cells may provide a factor(s) to hematopoietic cells that allow it to survive in medium lacking IL-3. At random, a retrotransposition may occur that provides a selective advantage to the hematopoietic cells. In the absence of the irradiated stromal cells, the hematopoietic cells are perhaps more likely to die and therefore are not available for a random retrotransposition event to occur. This model is to be distinguished from an active role in which the irradiated microenvironment would synthesize or activate a factor(s) that promotes retrotransposition.     

Composition and glycosaminoglycan metabolism of articular cartilage from habitually loaded and habitually unloaded sites

The uronic acid (proteoglycan, PG) content of cartilage from habitually unloaded sites of normal canine femoral condyles has been shown to be lower than that from habitually loaded regions, even though the glycosaminoglycan (GAG) synthesis is similar. We investigated whether the GAG degradation in unloaded cartilage would be greater than that in loaded cartilage, and we obtained comparative biochemical data concerning the PGs and organization of the extracellular matrix of normal loaded and unloaded cartilage. PG extractability (determined by sequential guanidinium chloride extracts of cartilage), percentage of PGs forming large aggregates, and hydrodynamic size of the PG monomers (determined by Sepharose 2B chromatography) were essentially the same in loaded and unloaded cartilage. As expected, the uronic acid content of unloaded cartilage was 20% lower than that of loaded cartilage (P less than 0.02), while the water and DNA contents of the 2 tissues were not statistically different. There was no difference in the rate of net 35SO4-GAG synthesis in organ cultures of loaded and unloaded cartilage. Moreover, there was no appreciable difference in the rates of 35SO4-GAG degradation of loaded and unloaded cartilage, as determined by 35SO4 pulse-chase studies. We have previously shown that selective cyclic compressive stresses applied in vitro to cartilage from loaded areas of canine femoral condyles may increase 35SO4-GAG synthesis. The present results suggest that the rates of GAG metabolism in loaded and unloaded cartilage under atmospheric pressure in vitro may not reflect the rates which exist in articular joints under compressive loads in vivo.     

Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

High glucose modifies heparansulphate synthesis by mouse glomerular epithelial cells

Background

Alterations in proteoglycan metabolism are involved in the pathogenesis of diabetic nephropathy. The aim of this study is to evaluate the effects of high glucose on proteoglycan production and to find a reliable in vitro model for the study of diabetic nephropathy.

Methods

A clone of mouse glomerular epithelial cells was cultured in media containing elevated (30 mmol) and physiological (5 mmol) glucose, or iso‐osmolar (30 mmol) mannitol concentrations. We evaluated the synthesis of ³⁵SO₄‐labeled molecules and the amount of proteoglycans by Sepharose CL6B and DEAE‐Sephacel chromatographies.

Results

A clear decrease (56%) in total cell‐layer proteoglycan synthesis was induced by 30 mmol glucose, in comparison with normal glucose. A reduction of 25% in medium associated proteoglycan synthesis was observed in high glucose cultured cells. After Sepharose CL6B, in cells cultured in high glucose, cell layer heparansulphate proteoglycan‐I (Kav 6B 0.04) synthesis was reduced by about 81%, heparansulphate proteoglycan‐II (Kav 6B 0.21) by about 87% and heparansulphate glycosaminoglycan (Kav 0.4–0.8) by about 91%, respectively. In mannitol‐incubated cells the reductions observed were less evident and not significantly different from those in normal glucose.

Conclusions

These results indicate that (1) glomerular epithelial cells play a central role in proteoglycan synthesis, (2) high glucose modifies the amount and influences the different species production of these macromolecules, while osmotic forces seem to be only partially involved in these effects, and (3) this cellular clone of glomerular epithelial cells can represent a reliable in vitro model for the study of the mechanisms involved in diabetic nephropathy. Copyright © 1999 John Wiley & Sons, Ltd.

     

Induction of growth alterations in factor-dependent hematopoietic progenitor cell lines by cocultivation with irradiated bone marrow stromal cell lines

We studied the production of hemopoietins by x-irradiated plateau-phase cultures of cloned marrow stromal cell lines derived from C3H/HeJ marrow, termed D2XRII and clone 11. The production of CSF in agar overlay of control or 10,000 rad irradiated stromal cultures was quantitated by induction of colonies in: overlaid fresh marrow, IL-3- dependent cell line 32D cl 3, or GM-CSF/IL-3-dependent cell lines FDCP- 1 or bg/bg cl 1. Conditioned media were tested for CSF by bioassay using fresh marrow cells, for M-CSF (CSF-1) by RIA, and for IL-3 and GM- CSF by microwell proliferation assay with 32D cl 3 and FDCP-1 cells, respectively. X-irradiation to doses that decreased CSF-1 to 40% of control levels (greater than 5,000 rad) resulted in a 30-fold increase in growth of FDCP-1 or bg/bg cl 1 cells in liquid co-culture or agar culture overlay with no detectable growth of 32D cl 3. The frequency of subculture of nonautocrine, factor independent (FI) variant clonal lines of FDCP-1 or bg/bg cl 1 cells was increased over 1000-fold by 15 weeks cocultivation with irradiated stromal cell cultures. FI subclonal lines formed tumors in syngeneic mice and contained no detectable poly A messenger RNA for GM-CSF or IL-3, and no elevation in c-myc, c-abl, c- src, or erb-B onc gene-specific messenger RNA compared to parent factor- dependent lines. These data indicate that x-irradiated plateau phase marrow stromal cells produce increased levels of cell contact-mediated biologically active hemopoietin(s) other than M-CSF, GM-CSF, or IL-3 and induce nonautocrine factor-independent malignant cell lines in vitro.     

Proteoglycans and vascular cell proliferation

The relationships that exist between proliferative states and proteoglycan (PG) synthesis have been examined in monkey aortic smooth muscle cells in culture. These cells were made quiescent in medium containing low serum (0.1%) and stimulated to divide by addition of either nanogram quantities or platelet-derived growth factor (PDGF) or medium containing 5% serum. Incorporation of [35S]sulfate into PG was increased during the first 24 h of growth stimulation, and this increase appeared to be principally in the large chondroitin sulfate proteoglycan (CSPG). Furthermore, addition of p-nitrophenyl beta-D-xyloside, which perturbs PG metabolism, inhibits cells from proliferating, suggesting that PG may be involved in facilitating cell division. Inhibition of cell proliferation by heparin and/or TGF-beta also causes elevated levels of 35S-sulfate incorporation into PG by these cells. These studies indicate that proteoglycan metabolism is modulated as a function of the growth of arterial smooth muscle cells; however, it is still uncertain whether PG play a direct or indirect role in the control of cell growth.     

Distribution of homing protein on hemopoietic stromal and progenitor cells.

Homing receptor is a membrane lectin of 110 kd molecular weight that recognizes galactosyl and mannosyl residues of an as yet unknown glycoconjugate. It is responsible for recognition and selective homing of hemopoietic progenitor cells after these cells are transplanted intravenously. Consequently, it is present on the surface of hemopoietic progenitor cells. To determine the distribution of this receptor on other cell types we performed standard binding assays in many cell types using galactosyl and mannosyl residues covalently bound to bovine serum albumin (G-BSA and M-BSA) as an index of homing receptor. BSA moiety was then labeled with 125I. The three cloned hemopoietic cell lines B6Sut, FDCP-1, and FDCP-mix all showed combined binding of G-BSA and M-BSA, whereas the lymphoid cell line L1210 showed only M-BSA, not G-BSA binding and, therefore, was considered to lack homing receptors. Similarly, stromal cell lines D2X and GB1/6 as well as primary marrow stroma (progenitor cell-depleted) did not show homing receptors as evidenced by combined binding of G-BSA and M-BSA. Nor did the nonhemopoietic stromal cell line Swiss 3T3 show the presence of homing receptors by these criteria. We conclude that homing receptors are distributed narrowly and are present on hemopoietic progenitor cells, but absent on hemopoietic stroma.     

Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor

Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.     

Canine stem cell factor (c-kit ligand) supports the survival of hematopoietic progenitors in long-term canine marrow culture.

The cDNA for canine stem cell factor (cSCF, c-kit ligand) was cloned and expressed in Escherichia coli. The recombinant protein (rcSCF), 165 amino acids in length, is very similar structurally to the soluble form of previously cloned and sequenced rodent and human SCFs. The biological effects of rcSCF were studied in a day-10 granulocyte-macrophage colony-forming unit (CFU-GM) clonogenic assay and in long-term liquid bone marrow culture of non-adherent hematopoietic cells in the absence of a stromal underlayer. Synergism in the stimulation of growth of CFU-GM was demonstrated between rcSCF and both recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and naturally occurring colony-stimulating activity present in the serum of a neutropenic dog. Alone, rcSCF was nonstimulatory for committed marrow precursors in methylcellulose cultures and had minimal effect on hematopoietic progenitor cell survival in stromaless, liquid cultures. When rcSCF was combined with phytohemagglutinin-stimulated canine lymphocyte-conditioned medium (PHA-LCM) or rh interleukin 6 (IL-6), with or without rhGM-CSF, CFU-GM survived for up to 5 weeks. The combination of rcSCF and rhGM-CSF, without rhIL-6, led to an early increase in CFU-GM in liquid cultures that declined more rapidly than in flasks that included rhIL-6. Survival of progenitor cells was negligible beyond 1 week in flasks with growth factor combinations lacking rcSCF. Sustained production of nonadherent cells in long-term cultures also was dependent on rcSCF in combination with canine PHA-LCM or recombinant human growth factors. It appears that rcSCF, like that from rodent and primate species, has the ability to influence the survival and proliferation of CFU-GM, and perhaps earlier progenitor cells, in hematopoietic tissues. In a long-term liquid culture system in which growth factor production by stromal cells is limited, rcSCF possesses a unique ability to maintain the viability of progenitor cells for up to 5 weeks.     

Distinct cartilage proteoglycan chromatographic elution patterns in advanced human hip osteoarthritis: correlations with histologic analysis

Articular cartilage explants were derived from 14 human osteoarthritic (OA) femoral heads. Newly synthesized, 35SO4 radiolabeled proteoglycans and endogenous uronic acid containing proteoglycans present in the extracellular matrix fell into one of 4 distinct patterns of elution on Sepharose CL-2B chromatography under associative conditions. There was a strong correlation between the elution patterns for newly synthesized and endogenous proteoglycans within a given hip specimen (p = 0.005). The proteoglycan elution pattern also strongly correlated when Day 1 and Day 20 explants were compared. The presence of a distinct subpopulation of hydrodynamically small proteoglycan (Kav, 0.6-0.9) correlated with a loss of both pericellular and extracellular matrix metachromasy in histologic sections of cartilage (p = 0.018). Our findings support the contention that different biochemical-histological subsets exist in "end-stage" hip OA.     

The heparin-binding proteins apolipoprotein E and lipoprotein lipase enhance cellular proteoglycan production

Apolipoprotein E (apoE) and lipoprotein lipase (LPL), key proteins in the regulation of lipoprotein metabolism, bind with high affinity to heparin and cell-surface heparan sulfate proteoglycan (HSPG). In the present study, we tested whether the expression of apoE or LPL would modulate proteoglycan (PG) metabolism in cells. Two apoE-expressing cells, macrophages and fibroblasts, and LPL-expressing Chinese hamster ovary (CHO) cells were used to study the effect of apoE and LPL on PG production. Cellular PGs were metabolically labeled with (35)[S]sulfate for 20 hours, and medium, pericellular PGs, and intracellular PGs were assessed. In all transfected cells, PG levels in the 3 pools increased 1.6- to 3-fold when compared with control cells. Initial PG production was assessed from the time of addition of radiolabeled sulfate; at 1 hour, there was no difference in PG synthesis by apoE-expressing cells when compared with control cells. After 1 hour, apoE-expressing cells had significantly greater production of PGs. Total production assessed with [(3)H]glucosamine was also increased. This was due to an increase in the length of the glycosaminoglycan chains. To assess whether the increase in PGs was due to a decrease in PG degradation, a pulse-chase experiment was performed. Loss of sulfate-labeled pericellular PGs was similar in apoE and control cells, but more labeled PGs appeared in the medium of the apoE-expressing cells. Addition of exogenous apoE and anti-human apoE antibody to both non-apoE-expressing and apoE-expressing cells did not alter PG production. Moreover, LPL addition did not alter cell-surface PG metabolism. These results show that enhanced gene expression of apoE and LPL increases cellular PG production. We postulate that such changes in vascular PGs can affect the atherogenic potential of arteries.     

Expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells regulates proliferation,differentiation, and maintenance of hematopoietic stem and progenitor cells

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.     

Expansion of CD34+ Cells on Telomerized Human Stromal Cells without Losing Erythroid-Differentiation Potential in a Serum-Free Condition

Erythropoiesis progresses from stem cell expansion on stromal cells through the formation of an erythroblastic island. Our aim was to assess the feasibility of using human stromal cells for erythroid production and differentiation. When cord blood CD34+ cells were cocultured with telomerized human stromal cells (hTERT-stromal cells) for 2 weeks, the CD34+ cells and burst-forming units-erythroid (BFU-E) significantly expanded, and a few hematopoietic cells transmigrated below the stromal layer. When nonadherent hematopoietic progenitor cells that had expanded above the hTERT-stromal cells (group B) were collected and subjected to our erythroid-differentiation protocol, they differentiated into erythroblasts with a slight hemoglobin synthesis. When the few hematopoietic cells that had transmigrated below the stromal layer were expanded for an additional 2 to 6 weeks, they exhibited a cobblestone-like appearance, and a large amount of BFU-E clambered weekly from the underside of the stromal layer to above the stromal layer (group C). When the hematopoietic progenitor cells in group C were subjected to the erythroid-differentiation protocol, large numbers of mature erythroblasts (more than 300,000 times the initial CD34+ cell number) were produced. Our hTERT-stromal expansion protocol may contribute to the construction of a system for large-scale, long-term production of erythroid cells.     

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1

Background

Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs.

Design and Methods

We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells.

Results

We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor.

Conclusions

Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.