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(Circulation. 2003;107:2078.)
© 2003 American Heart Association, Inc.

Brief Rapid Communications

Isolation of Bone Marrow Stromal Cell–Derived Smooth Muscle Cells by a Human SM22 Promoter

In Vitro Differentiation of Putative Smooth Muscle Progenitor Cells of Bone Marrow

Yuji Kashiwakura, MD; Youichi Katoh, MD, PhD; Kenji Tamayose, MD, PhD; Hakuoh Konishi, MD, PhD; Norihide Takaya, MD, PhD; Senji Yuhara, MD; Masanori Yamada, PhD; Koichi Sugimoto, MD, PhD; Hiroyuki Daida, MD, PhD

From the Departments of Cardiology (Y. Kashiwakura, Y. Katoh, H.K., N.T., H.D.), Hematology (K.T., K.S.), Gynecology (S.Y.), and Neurology (M.Y.), Juntendo University School of Medicine, Tokyo, Japan.

Correspondence to Youichi Katoh, MD, PhD, Department of Cardiology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail Katoyo{at}med.juntendo.ac.jp

	Abstract

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Background— Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs.

Methods and Results— To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22 promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-ß but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-ß signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-ß.

Conclusions— We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Key Words: bone marrow • stromal cells • stem cells • cell differentiation • muscle, smooth

	Introduction

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A better understanding of smooth muscle development and differentiation will help us more clearly define the vascular disease process. Smooth muscle cells (SMCs) in the large vessels close to the heart and pericytes in the forebrain are derived from the neural crest,¹ as well as embryonic carcinoma cells^2,3 and embryonic stem cells.⁴ A very recent report showed that bone marrow hematopoietic stem cells (HSCs) differentiate into SMCs in cocultures with primary rat aortic SMCs.⁵ Bone marrow stromal cells (BMSCs), the adhesion cell fractions of bone marrow mononuclear cells, may also have the potential to differentiate into SMCs.^6–8 Although there have been data suggesting the coexistence of a smooth muscle lineage with several other lineages in bulk-BMSCs,^6–8 there have been few studies closely following cell development of smooth muscle lineage cells among BMSCs.

To investigate the possible existence of a cell population committed to the SMC lineage among adhesion cells of bone marrow, we tried to detect and follow the in vitro differentiation of such a cell type by a tissue-specific promoter sorting method. We elucidated the process of SMC development among BMSCs by investigating the isolated BMSC-derived SMCs for their morphology and their expression of SMC-specific mRNA and protein.

	Methods

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Cell Cultures and Plasmid Construction
Mononuclear cells obtained by the Ficoll-Paque protocol⁹ from murine (C57BL6 mouse) bone marrow (2x10⁵ nuclear cells/mL) were cultured in Dexter-type condition.¹⁰ Bone marrow–derived adhesion cells appeared within a week (early phase adhesion cells, E-ad cells) and gradually grew into BMSCs over the next 3 to 7 weeks.

A 480-base fragment of the human SM22 promoter that works specifically in smooth muscle lineage cells¹¹ was obtained by PCR with the following primers: forward 5'-GGATCCCATGTCCCATCAGA-3' and reverse 5'-GGGGCGCTGGCTGGGTGAGG-3'. The fragment was integrated into a promoterless GFP vector (pd2EGFP, Clontec). By the lipofection method (Invitrogen), the plasmid was transfected to the early phase adhesion cells on the 5th day after seeding of the mononuclear cells. Using cloning cylinders (Sigma), 15 individual clones were successfully obtained by G418 (500 µg/mL, GIBCO) selection.

Immunocytochemistry
Immunocytochemistry was performed using antibodies recognizing platelet-derived growth factor-ß receptor (PDGF-ß, Santa Cruz, diluted 1: 250), Flk-1 (Santa Cruz, diluted 1:200), smooth muscle myosin heavy chain (SM) embryo, (SMemb, marker protein of immature SMCs, diluted 1:400), calponin h1 (Santa Cruz, diluted 1:100), -smooth muscle actin (-SM actin, Biomega, diluted 1:200), and as well as smooth muscle myosin heavy chain-1 (SM-1, marker protein of mature SMC,¹² diluted 1:400). Both SMemb and SM-1 antibodies were kindly provided by Dr M. Periasamy (Columbus, Ohio).

Reverse-Transcriptase Polymerase Chain Reaction
Mouse aorta tissue total RNA was extracted using the guanidinium thiocyanate method. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed using Promega’s Access RT-PCR kit (Promega). The following primers for mouse SM1 and SM2 were designed to amplify 251-bp and 341-bp cDNA products, respectively, and their sequence are as following: SM1 forward 5'-CTCAAGAGCAAACTCAGGAG-3', SM2 forward 5'-CTCAA-GAGCAAACTCAGAGG-3', SM1 reverse 5'-TCTGTGAC-TTGAGAACGAAT-3', and SM2 reverse 5'-ACACCCTTTGTGC-AGGGCTGA-3'.

Western Blot Analysis
Western blot analysis was performed using -SM actin antibodies (diluted 1:200, 3 µg of protein), calponin h1 antibodies (diluted 1:200, 10 µg of protein), and SM1 antibodies (diluted 1:300, 20 µg of protein from mouse aorta and brain and 70 µg of protein from the other samples were subjected.).

	Results

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On the basis of the transfection efficiency calculated from CMV promoter-GFP promoterless (pd2EGFP) vector transfections into the early phase adhesion cells (E-ad cells), we determined that about 1% to 3% cells of the E-ad cells expressed GFP stably. We recognized 20 clusters of Ad(G) cells that subsequently showed colony formation expressing SM22-driven GFP. The following data are from clone number 7 (CL-7), a representative clone among the 15 individual clones. Figure 1A shows the course used to acquire samples.

View larger version (40K): [in this window] [in a new window]   Figure 1. A, Schema of the course for acquiring samples such as Ad(G) cells, smooth muscle lineage individual clones, and bulk-BMSCs. Briefly, bone marrow early phase adhesion cells (E-ad cells) from bone marrow mononuclear cells (MNCs) were transfected with a human SM22 promoter/GFP construct. GFP-positive cells [Ad(G) cells] were first detectable 5 days after the transfection. At that time, they expressed neither mature (calponin) nor immature (SMemb) SMC-specific proteins (dashed arrow). Using G418 selection, they were grown into 15 individual clones. Among all clones generated, there was little difference in phenotypes or in the expression patterns of SMC-specific protein and mRNA. Clones expressed calponin, SM-1 (dashed arrow). We chose clone number 7 (CL-7) as a representative clone. Bulk-BMSCs were plainly cultured. B, Light microscopic detection of Ad(G) cells. Five days after the transfection of the promoter/GFP construct into E-ad cells, GFP were first detectable in a cell population [Ad(G) cells, panel a and b, arrow]. Magnification x40. C, Confocal photomicrographs of Ad(G) cells. Ad(G) cells (panels a and b) were stained by PDGF-ß (panel c) antibodies but not by Flk-1 (panel d) antibodies. Magnification x100.

Ad(G) Cells Were Stained by PDGF-ß, but not Flk-1, SMemb, or Calponin Antibodies
Five days after the transfection of the promoter/GFP construct into the E-ad cells, GFP was first detectable in a cell population [Ad(G) cells, Figure 1B, a and b, arrow]. As shown in Figure 1C, Ad(G) cells (panel a and b) were stained by PDGF-ß (panel c) antibodies but not by Flk-1 (panel d) antibodies. On the other hand, Ad(G) cells were not stained by neither SMemb nor calponin antibodies at that time (data not shown).

Individual Clones That Generated From Ad(G) Cells Were Positive for Antibodies Against SMC-Specific Antibodies
CL-7 cells were thoroughly stained by the three SMC-specific antibodies (-SM actin, calponin, and SM1; Figure 2A).

View larger version (55K): [in this window] [in a new window]   Figure 2. A, Confocal photomicrographs of CL-7 stained by -SM actin, calponin, and SM-1 antibodies. Three clusters of CL-7 cells (panels a, b, and c) were clearly stained by the SMC-specific proteins’ antibodies that were reacted with Texas-red second antibody (panels d, e, and f). Magnification x100. B, RT-PCR analysis of SM1 and SM2 mRNA transcripts in clones. Two-microgram portions of total RNA from CL-7 cells in various phases (0 days, 3 days, 6 days, and more than 9 days after the first passage of individual clones) were used to amplify SM1 (251-bp) and SM2 (341-bp) fragments. Both SM1 and SM2 cDNA products in the same phase were applied to the same lane of 2% agarose gel. Total RNA extracted from mouse aorta was used as positive control. C, Western blot analysis in mouse aorta, E-ad cells, CL-7 cells, bulk-bone marrow stromal cells (Bulk-Str), and mouse brain. Using -SM actin, calponin, and SM1 antibodies, blotting analyses were performed. An -tubulin antibody was used as an internal control (data not shown). Proteins extracted from early phase adhesion cells (E-ad cells) containing Ad(G) cells were subjected to the lane of E-ad. CL-7 expressed calponin and SM1 proteins, whereas E-ad cells containing Ad(G) cells expressed neither calponin nor SM1.

mRNA of Both SM1 and SM2 Were Expressed in CL-7 Cells
As shown in Figure 2B, the RT-PCR products corresponding to SM1 and SM2 mRNA transcripts migrated as 251-bp and 351-bp bands, respectively. Expression of the SM1 was constant in CL-7 cells during the cultures, whereas the expressions of SM2 mRNA were only weakly detected and observable only during limited periods, such as within 4 to 8 days after the first passage of individual clones. These results suggested that SM2, a marker for mature SMCs, was easily downregulated in the clones during the culture, as shown in an early study.¹³

Clones Contained Calponin and SM1 Proteins
As shown in Figure 2C, calponin and SM1 protein were expressed in all clones to some extent, whereas the early phase adhesion cells (E-ad cells) including Ad(G) cells expressed no SMC-specific proteins.

	Discussion

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A recent study demonstrated that circulating smooth muscle progenitor cells facilitate the development of proliferative diseases such as graft vasculopathy.¹⁴ Moreover, intimal SMCs in graft arterial disease can originate from recipient bone marrow cells.¹⁵ Under in vitro conditions, bone marrow is fractionated not only to floating cell types such as hematopoietic stem cells (HSCs), but also to adhesion cell types such as BMSCs. Bone marrow HSCs (c-Kit+, Sca-1+, Lin-) can differentiate into SMCs in cocultures with primary rat aortic SMCs.⁵ On the other hand, there have been few studies closely following cell differentiation of smooth muscle lineage among BMSCs. The present study demonstrated that there was a unique cell population fated to become SMCs among BMSCs. They expressed no SMC-specific marker proteins but later came to exhibit phenotypes consistent with SMC lineage, suggesting that they might at least have partly progenitor properties. Moreover, Ad(G) cells expressed PDGF-ß. PDGF-ß, one of the most important receptors contributing to the recruitment of SMC progenitor in angiogenesis, has been suggested to be crucial for differentiation of mesenchymal-derived SMCs.^16,17 Although PDGF-ß is not a definitive marker for smooth muscle progenitors, Ad(G) cells might be putative mesenchymal smooth muscle progenitors.

BMSCs have been investigated as vehicles for both cell and gene therapy^18,19 in the past few years. These cells are relatively easy to isolate from the small aspirates of bone marrow that can be obtained under local anesthesia. They are also relatively easy to expand in cell culture and to transfect with exogenous genes. For these reasons, BMSCs seem to have several advantages over HSCs for use in gene therapy. Large volumes of bone marrow are required for the isolation of adequate numbers of HSCs, and the cells are difficult to expand in culture. Thus, there is much to be gained by establishing BMSCs as a source of large amounts of SMCs for potential use in cell and gene therapy.

Although there is accumulating evidence that BMSCs have many characteristics of mesenchymal stem cells that can differentiate into SMCs,^6–8 there have been few studies closely following cell development of smooth muscle lineage among BMSCs or establishing BMSCs as a source of large amounts of SMCs. This is caused by the difficulty isolating smooth muscle lineage cells from the whole of bulk-BMSCs because smooth muscle lineage cells coexist with several other lineage cells among bulk-BMSCs. The present tissue-specific promoter sorting method provides unique opportunities to isolate SMC lineage from indistinct combinations of many cell types among BMSCs. Moreover, the BMSC-derived SMCs obtained in this study may have more self-expansion abilities than HSCs. For this reason, they have good prospects to become a useful material for cell and gene therapy.

In conclusion, we isolated and observed a unique cell population fated to become SMCs among BMSCs. These cells expressed PDGF-ß but neither mature nor immature SMC-specific proteins. By following the longitudinal transition of their phenotypes, we found that they eventually differentiated into SMCs possessing a certain level of self-expansion abilities. Although further studies are necessary to define these cells, our data suggest that Ad(G) cells may be putative mesenchymal smooth muscle progenitors expressing PDGF-ß. The sorting system reported here has the potential to provide large amounts of BMSC-derived SMCs of potential use in cell and gene therapy.

Note Added in Proof
During the review process of this article, Simper et al²⁰ reported that peripheral hematopoietic mononuclear cells differentiate in culture into smooth muscle cells.

	Acknowledgments

 
This study was supported in part by a High Technology Research Center Grant and Grant-in Aid for Scientific Research (C) (to Dr Katoh) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Yasue Takahashi for expert technical assistance.

	Footnotes

 
Figures A through G can be found in the online-only Data Supplement at http://www.circulationaha.org.

Received January 30, 2003; revision received March 12, 2003; accepted March 13, 2003.

	References

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 107/16/2078    most recent 01.CIR.0000070082.64414.B5v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kashiwakura, Y. Articles by Daida, H. Search for Related Content PubMed PubMed Citation Articles by Kashiwakura, Y. Articles by Daida, H. Related Collections Developmental biology Smooth muscle proliferation and differentiation