Expression of Bone Morphogenetic Proteins in Stromal Cells from Human Bone Marrow Long-term Culture
Department of Anatomy, Medical School University of Zagreb (SM,VK,FB,PS,LG,SV); Clinical Institute for Laboratory Diagnosis, Clinical Hospital Center Zagreb (SM,DB); and Departments of Internal Medicine (NB,BL) and Pathology (JJ-R), Clinical Hospital Center Zagreb, Zagreb, Croatia
Correspondence to: Slobodan Vukicevic, MD, PhD, Dept. of Anatomy, Medical School University of Zagreb, Salata 11, Croatia. E-mail: vukicev{at}mef.hr
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Summary |
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Key Words: BMPs bone marrow stromal cells hemopoiesis long-term culture
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Introduction |
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The regulation of hemopoiesis is a complex process, which requires signaling among stromal cells, stem cells, and progenitor cells. Members of TGF-ß family signal through two types of serine/threonin kinase receptors and phylogeneticaly highly conserved intracellular transducer molecules SMADs (Korchynsky and ten Dijke 2002; Miyazawa et al. 2002
). Various mechanisms precisely regulate BMP signals by positive regulation and negative feedback, both important for gradient formation, which is needed for proper development (Jonsson et al. 1997
; Miyazono 2000
; Miyazono et al. 2001
; Korchynsky and ten Dijke 2002
; Martinovic et al. 2002
). Moreover, signaling by SMADs is often modulated by crosstalk with other signaling pathways which gives this superfamily a broad array of biological activities (Miyazono et al. 2001
).
It is unknown whether BMPs are expressed in adult and differentiated stromal cell layers required to support hemopoiesis and eventually to play a role in regulation of hemopoietic stem cell differentiation. We report that long-term cultures produce BMPs that are required for the maintenance of hemopoiesis.
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Materials and Methods |
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Immunohistochemistry
Cells were seeded in eight-well chamber slides at a concentration of 12.5 x 104/ml (5 x 104/well) and incubated in Myelocult H5100 medium for 5 weeks. Cells were fixed in 4% paraformaldehyde in PBS for 5 min, washed three times in PBS, immersed in methanol for 1 min, treated with 3% H2O2 for 5 min, and washed in PBS. Immunostaining was performed using BMP-3 (Vukicevic et al. 1994a), BMP-4 (Santa Cruz Biotechnology; Santa Cruz, CA), and BMP-7 antibody (Vukicevic et al. 1994b
) with a biotinylated secondary antibody (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and horseradish peroxidase-conjugated streptavidin (Vector). Parallel cultures were stained with rabbit anti-human lysozyme, mouse anti-human CD31, mouse anti-human factor VIII, mouse anti-human CD68, rabbit anti-cow S100, rabbit anti-calf alkaline phosphatase, and rabbit anti-vimentin antibodies (DAKO; Glostrup, Denmark). For negative controls the primary antibody was replaced by BSA or the secondary antibody alone, and skin and bone marrow tissue samples were used as positive controls.
Reverse Transcription PCR Analysis
Total RNA was extracted from freshly isolated bone marrow samples, purified peripheral lymphocytes, and bone marrow samples after 1, 3, 5, and 8 weeks in culture using the guanidine thiocyanate/acid phenol method as indicated by the manufacturer (TRIzol reagent; Gibco BRL, Grand Island, NY). Contaminating genomic DNA was removed with RNase-free DNase (Gibco BRL). cDNA was synthesized with Superscript II RNase H-Reverse Transcriptase as indicated by the manufacturer (Gibco BRL). PCR was performed in the PE GeneAmp 2400 thermal cycler (Perkin-Elmer; Norwalk, CT) using the following primers: GAPDH (5' ACC ACA GTC CAT GCC ATC AC, 3' TCC ACC ACC CTG TTG CTG TA); BMP-2 (5' CAG AGA CCC ACC CCC AGC A, 3' CTG TTT GTG TTT GGC TTG AC); BMP-3 (5' TTT CTC TCC TCC CAC ACC, 3' CAA TCT GAC ATC GCT AAC C); BMP-4 (5' TTC CTG GTA ACC GAA TGC T, 3' GGG GCT TCA TAA CCT CAT A); BMP-5 (5' ACG GAA CCA CGA AAG ACG, 3' GCC AAC CCA CAT CTA AAG C); BMP-6 (5' GCA GAA GGA GAT CTT GTC GG, 3' AGC TGA AGC CCC ATG TTA TG); BMP-7 (5' TGG CGT TCA TGT AGG AGT TCA G, 3' ACG CTT CGA CAA TGA GAC GTT C); ALK-2 (5' TGG AAG ATG AGG AGC CCA AGG T, 3' GAA GTT CTG CGA TCC AGG GAA G); ALK-3 (5' CTG CTG CGC TCA TTT ATC, 3' ACC ATC GGA GGA GAA ACT); ALK-6 (5' AAG TTA CGC CCC TCA TTC, 3' TGA TGT CTT TTG CTC TGC); SMAD1 (5' CGA ATG CCT TAG TGA CAG, 3' GAG GTG AAC CCA TTT GAG); SMAD4 (5' AGG TGA AGG TGA TGT TTG, 3' GCT ATT CCA CCT ACT GAT) and SMAD5 (5' AGA TAT GGG GTT CAG AGG, 3' TGT TGG TGG AGA GGT GTA) (Invitrogen Life Technologies; Carlsbad, CA).
Reactions included 5 µl 10x buffer (Promega; Madison, WI), 3 µl MgCl2 (Promega), 1 µl dNTP, 1 µl 3' primer, 1 µl 5' primer, 0.5 µl Taq polymerase (Promega), and 1 µl cDNA. After initial denaturation at 94C for 5 min, 32 to 40 cycles of amplification were completed by denaturation for 40 sec at 94C, annealing at temperature specified for each pair of primers for 40 sec, and extension for 60 sec at 72C. To compare the relative quantity of the RT-PCR reactions, the transcription level of GAPDH, a "housekeeping" gene, was used as control. Reactions without cDNA were used as negative control and kidney cDNA as a positive control. Results were visualized by gel electrophoresis in 1% agarose (Seakem GTG; Bioproducts, Rockland, MA) in TAE buffer (Tris-HCl, acetic acid, EDTA, pH 8.0) and stained with ethidium bromide (Sigma-Aldrich; St Louis, MO). Reactions were repeated at least twice.
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Results |
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Expression of BMP Receptors and Signaling Molecules by BM Stromal Cells
Figure 5
shows the RT-PCR results obtained using specific primers for the BMP type I receptor, activin-like kinase-3, which was expressed strongly during the first few weeks of culture in all samples, as well as in bone marrow and peripheral lymphocytes (Figure 5). Similar results were obtained for human ALK-6 primers, except for peripheral lymphocytes. In contrast, no expression of ALK-2 transcripts in human bone marrow stromal cells was found (Figure 5).
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Discussion |
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Differentiation of hemopoietic stem cells into lineages depends on stromal cells and their specific signals. It has been shown that stromal cell layer possesses the ability to support B-lymphopoiesis for up to 9 months (McGinnes et al. 1991). Treatment of isolated stem cell populations with soluble BMPs induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Bhatia et al. 1999
). Treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation and supported the maintenance of the primitive CD34+CD38 phenotype. Low concentrations of BMP-4 induced proliferation and differentiation of CD34+CD38Lin cells, while at higher concentrations it extended the time of repopulation capacity in ex vivo culture (Bhatia et al. 1999
). BMP-4 activates ectodermal and mesodermal markers in human embryonic stem cells (Schuldiner et al. 2000
). Furthermore, BMP-4 was shown to stimulate expression of GATA-2, a DNA-binding protein that regulates hemopoiesis-specific gene transcription in Xenopus laevis (Maeno et al. 1996
). Exogenous BMP-6 was found to block the VLA4/VCAM-1 adhesion pathway mediating the adhesion of primitive hemopoietic stem cell to marrow stroma, thus enabling mobilization of CD34+ cells, which may have a potential therapeutic use in patients with myeloid leukemia lacking BMP expression (Ahmed et al. 2001
). BMP-6 significantly reduces IL-6 and IL-11 production from the marrow stroma (Ahmed et al. 2001
) and stimulates their common receptor gp130, implying its possible role as a therapeutic agent in multiple myeloma. It also promotes chondrogenesis in a subpopulation of small and rapidly self-renewing marrow stromal cells (Sekiya et al. 2001
,2002
). Exogenously added BMPs may evoke elaboration and release of hemopoietic cells from the bone marrow. However, endogenously produced members are responsible for differentiation of hemopoietic cells and possibly for determination of the mesenchymal cell fate and their commitment to the specific lineage. Recently, it was reported that members of TGF-ß family, their receptors, and their second messengers may play a role in the development of certain types of neoplasms (Miyazono et al. 2003
). Loss of functional cell surface TGF-ßR type I correlates with insensitivity to TGF-ß in chronic lymphatic leukemia (DeCoteau et al. 1997
). The current use of BMPs in reconstruction of the skeleton provides us with hope that BMPs might have therapeutic indications in patients with acute leukemias and lymphomas.
The results of this study contribute to understanding of the mechanism that enables the bone marrow stromal cell population to support production and maintenance of hemopoietic cells.
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Footnotes |
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