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Expression of Bone Morphogenetic Proteins in Stromal Cells from Human Bone Marrow Long-term Culture

Snjezana Martinovic, Sanja Mazic, Veronika Kisic, Nikolina Basic, Jasminka Jakic-Razumovic, Fran Borovecki, Drago Batinic, Petra Simic, Lovorka Grgurevic, Boris Labar and Slobodan Vukicevic

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

	Summary

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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. (J Histochem Cytochem 52:1159–1167, 2004)

Key Words: BMPs • bone marrow stromal cells • hemopoiesis • long-term culture

	Introduction

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INVESTIGATIONS OF MOLECULES involved in regulation of human hemopoietic stem cells have focused mainly on cytokines, of which very few are known to have a direct effect on stem cells (Metcalf 1993,1998; Ogawa 1993). Experimental work using developmental systems showed that tissue differentiating from the ventral mesoderm contain stem cells capable of multilineage hemopoietic differentiation (Rollins-Smith and Blair 1990; Kelley et al. 1994; Yoder et al. 1994,1997; Palis et al. 1995; Medvinsky and Dzierzak 1996; Choi et al. 1998; Huber and Zon 1998). Analyses have revealed the growth factors responsible for hemopoietic differentiation of ventral mesodermal precursors and have confirmed that the majority of them were members of the transforming growth factor beta (TGF-ß) superfamily of secreted polypeptides (Mishina et al. 1995; Winnier et al. 1995; Maeno et al. 1996; Garbe et al. 1997; Kishimoto et al. 1997). In addition to TGF-ß as the prototype, this family consists of activins, inhibins, and bone morphogenetic proteins (BMPs). The BMP subfamily of signaling polypeptides exerts a wide range of biological responses with direct effects on cell growth, proliferation, differentiation, and apoptosis. Their role is highly emphasized in patterning during embryonic development as well as in morphogenesis of different tissues and organs (Vukicevic et al. 1994a,b; Helder et al. 1995; Vukicevic et al. 1996; Dudley and Robertson 1997; Massague 1998; Whitman 1998; Martinovic et al. 2002). In adult tissues, these proteins maintain tissue homeostasis (Vukicevic et al. 1998; Martinovic et al. 2002) and their genes can be regulated by other cytokines and growth factors (Paralkar et al. 2002). The genes that encode bone morphogenetic proteins are part of developmental regulatory circuits both upstream and downstream of homeobox-containing genes (Iimura et al. 1994; Reuter et al. 1990; Mead et al. 1996). The homeobox genes are a family of transcription factors involved in determining cell fate during embryogenesis (Krumlauf 1994), and in the control of lineage specificity during hemopoiesis (Mathews et al. 1991; Lawrence et al. 1993; Giampaolo et al. 1994). Therefore, BMPs are candidates for regulators of hemopoietic differentiation and function in mature blood cells. Recent studies have confirmed the effect of BMPs on highly primitive as well as highly differentiated hemopoietic cells (Bhatia et al. 1999; Detmer et al. 1999). One of these proteins, BMP-4, was found to be a potent ventralizing factor, which can induce hemopoietic tissue in Xenopus and mice (Harland 1994; Johansson and Wiles 1995). BMP-4 and transcriptional factor GATA-2 act in adjacent germ layers and participate in blood cell formation during embryogenesis (Maeno et al. 1996). Furthermore, subcutaneous implantation of BMP-2 and BMP-7 into rats can induce osteogenesis and the hemopoietic microenvironment that supports the growth of stem cells (Rath and Reddi 1979; An et al. 1996). Many TGF-ß superfamily members play multiple roles in hemopoiesis. Activin A is produced by activated peripheral blood monocytes (Eramaa et al. 1992; Shao et al. 1992) and promotes erythroid differentiation (Mizuguchi et al. 1993). Different isoforms of TGF-ß have both stimulatory and inhibitory effects on hemopoietic progenitor cells (Ottmann and Pelus 1988; Jacobsen et al. 1991; Van Ranst et al. 1996). BMP-9 acts as a hemopoietic hormone (Ploemacher et al. 1999). The inhibition of BMP signal transduction in post-gastrulation mesoderm results in specific defects in primitive erythropoiesis of Xenopus embryos (Schmerer and Evans 2003). In adult vertebrates, BMPs are expressed in variety of tissues, including bone marrow (Vukicevic et al. 1990,1994a,b; Helder et al. 1995), and are essential in bone remodeling and growth (Vukicevic et al. 1993; Martinovic et al. 2002). Most of the studies exploring the potential role of BMPs in hemopoietic tissue have relied on model systems involving lower organisms and in vitro systems (Zon 1995; Mead et al. 1996). Recently, two studies using more biologically relevant human hemopoietic cell models have been published. Highly purified primitive human hemopoietic cells obtained from a suspension of cord blood expressed BMP type I receptors and signaling transducer molecules. They responded to exogenously added BMP-2, -4, and -7, which regulated their proliferation and differentiation with the direct effect of stem cell survival (Bhatia et al. 1999). Another study showed that normal adult hemopoietic cell lines express BMP genes with lineage-restricted patterns of expression, with BMP-4 restricted to T-lymphoid lineage, BMP-7 to lymphoid, PLAB (placental bone morphogenetic protein) to macrophage/monocyte lineage, and GDF-1 to myeloid lineage (Detmer et al. 1999). It was also shown that individual BMPs act in synergy with hemopoietic cytokines to induce differentiation of different hemopoietic progenitors (Detmer and Walker 2002), forming a part of the cytokine network regulating their development. Moreover, apart from fetal circulation, liver, and bone marrow, it was recently demonstrated that multiple blood lineages could be induced from human skeletal muscle or neural tissue by the combination of cytokines, BMP-4, and erythropoietin (Jay et al. 2002). Furthermore, BMP-4 inhibits proliferation and induces apoptosis of multiple myeloma cell lines as well as freshly isolated cells from patients with multiple myeloma (Hjertner et al. 2001). When added to the culture of rhesus monkey embryonic stem cells, it increases the numbers of hemopoietic clusters that contain hemopoietic-like CD34+ cells with morphological features of undifferentiated blasts (Li et al. 2001).

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.

	Materials and Methods

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Cell Culture
Normal human bone marrow specimens for long-term culture were obtained from healthy donors by aspiration of the posterior iliac crest during the standard procedure for allogenic bone marrow transplantation. The procedures followed were in accordance with the Helsinki Declaration of 1975 and approved by the Ethical Committee of Medical School University of Zagreb, Croatia. Long-term cultures of human hemopoietic cells were established according to Eaves et al. (1991). Briefly, mononuclear cells were separated on a Ficoll density gradient (Amersham Pharmacia Biotech; Uppsala, Sweden) and cultivated in Myelocult H5100 medium (StemCell Technologies; Vancouver, Canada) with freshly prepared and filter-sterilized hydrocortisone sodium succinate (final concentration of 10^–6 M). The cultures were kept for 4 days at 37C, and after the initiation of the adherent stromal layer were transferred to 33C in a humidified atmosphere with 5% CO₂. Half-media were changed once weekly and cultures were maintained up to 8 weeks. Short-term cultures of clonogenic progenitors were prepared at every half-medium change. Cells were seeded in semisolid Methocult H4433 medium (StemCell Technologies) at a concentration of 50,000 cells/well and were cultivated for 14 days. Human clonogenic progenitors were scored according to standard criteria (Rogulj et al. 1999).

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.

	Results

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Identity and Clonogenicity of Long-term Cultured Bone Marrow Stromal Cells
The identity of heterogeneous stromal cells was confirmed by immunostaining with commonly used markers: factor VIII and CD31 for endothelial cells; lysozyme, CD68, and S100 for macrophages; vimentin for fibroblasts; and alkaline phosphatase for committed preosteoblasts (Figure 1) .

View larger version (136K): [in this window] [in a new window]   Figure 1 Immunohistochemical staining of human bone marrow stromal cells. Cells were cultured for 5 weeks and stained as described in Materials and Methods for vimentin (A), human CD31 (B), human CD68 (C), and alkaline phosphatase (D). Control staining without secondary antibody (E). Magnifications: A,E x400; B,C,D x200.

View larger version (74K): [in this window] [in a new window]   Figure 2 Short-term culture of clonogenic progenitors. Cells were seeded at 50,000 cells/well in semisolid Methocult medium at medium change after 4, 5, 6, and 7 weeks in culture, cultivated for 14 days, and number of clonogenic progenitors was scored according to standard criteria. Magnification x150.

View larger version (113K): [in this window] [in a new window]   Figure 3 Expression of BMPs in stromal cells from human bone marrow long-term culture. Cells were cultured and gene expression analyses were done as described in Materials and Methods. RNA was isolated from freshly isolated bone marrow (BM), peripheral lymphocytes (Ly), or cultures at designated time points (1, 3, 5, 8 weeks), cDNA was synthesized and semiquantitative RT-PCR performed as described.

View larger version (121K): [in this window] [in a new window]   Figure 4 Immunostaining of human bone marrow stromal cells. Cells were cultured for 5 weeks (A) and stained as described in Materials and Methods using BMP-3- (B), BMP-4- (C), and BMP-7-specific antibodies (D). Magnification x200.

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).

View larger version (66K): [in this window] [in a new window]   Figure 5 Expression of type I receptors (activin-like kinases 2, 3, and 6) in stromal cells from human bone marrow long-term culture. Cells were cultured and gene expression analyses were done as described in Materials and Methods. RNA was isolated from freshly isolated bone marrow (BM), peripheral lymphocytes (Ly), or cultures at designated time points (1, 3, 5, 8 weeks), cDNA was synthesized and semiquantitative RT-PCR performed as described.

View larger version (59K): [in this window] [in a new window]   Figure 6 Expression of SMAD transducers in stromal cells from human bone marrow long-term culture. Cells were cultured and gene expression analyses were done as described in Materials and Methods. RNA was isolated from freshly isolated bone marrow (BM), peripheral lymphocytes (Ly), or cultures at designated time points (1, 3, 5, 8 weeks). cDNA was synthesized and semiquantitative RT-PCR performed as described.

	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+CD38–Lin– 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.

	Footnotes

 
Received for publication January 22, 2004; accepted April 29, 2004

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