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Hiroshi Kubo^1,, Masakazu Shimizu^1,, Yuji Taya², Takeshi Kawamoto¹, Masahiko Michida³, Emi Kaneko¹, Akira Igarashi¹, Masahiro Nishimura⁴, Kazumi Segoshi⁴, Yoshihito Shimazu², Koichiro Tsuji⁵, Takaaki Aoba² and Yukio Kato^1,*

¹ Department of Dental and Medical Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
² Department of Pathology, Nippon Dental University, Tokyo, Japan
³ Department of Orthodontics and Craniofacial Developmental Biology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
⁴ Prosthetic Dentistry, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
⁵ Two Cells Co. Ltd, Hiroshima, Japan

	Abstract

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Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes—including nine transcription factor genes —using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis- or adipogenesis-induction medium, or 48–72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self-renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic- and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules—including GATA6, TRPC4, FLG and TGM2—revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.

	Introduction

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Bone marrow-derived mesenchymal stem cells (MSC) adhere to the culture surface and can be separated from the majority of non-adherent bone marrow cells: They proliferate in the undifferentiated state, and then differentiate into osteoblasts, chondrocytes or adipocytes in appropriate differentiation-induction media (Pittenger et al. 1999; Caplan & Bruder 2001; Sekiya et al. 2002). Furthermore, transplantation of ex vivo expanded MSC enhanced regeneration of bone, cartilage, periodontal tissue, heart and brain, etc. in animal models and clinical studies (Horwitz et al. 1999; Kopen et al. 1999; Deans & Moseley 2000; Quarto et al. 2001; Wakitani et al. 2002; Kawaguchi et al. 2004; Yamada et al. 2004; Laflamme & Murry 2005; Hao et al. 2006; Miyahara et al. 2006). Thus, ex vivo expanded MSC have been used in a vast number of basic studies and numerous clinical trials. Nonetheless, the molecular signature and in vivo distribution status of bone marrow MSC remain unclear (Tremain et al. 2001; Jia et al. 2002; Silva et al. 2003; Wieczorek et al. 2003).

Several DNA microarray analyses have compared gene expression profiles of MSC and fibroblasts (Brendel et al. 2005; Ishii et al. 2005; Wagner et al. 2005), or MSC and some differentiated cells (Doi et al. 2002; Wieczorek et al. 2003; Hung et al. 2004; Monticone et al. 2004; Salasznyk et al. 2005; Song et al. 2006), but no dependable conclusions have been reached. As we would expect MSC-characteristic genes to be suppressed in any differentiated cells and skin fibroblasts, gene filtering—using bone marrow MSC, various differentiated cells and skin or gum fibroblasts —may enable selection of appropriate MSC-characteristic genes along with removal of unrelated genes. In addition, an MSC-related profile should show a similarity to that of other MSC (Kim et al. 2006), so we assumed a comparison of the profiles of bone marrow MSC and synovial fibroblasts/MSC—which also have osteogenic, chondrogenic and adipogenic potential (De Bari et al. 2001; Lee et al. 2004; Sakaguchi et al. 2005)—would be worthwhile. It would also be interesting to determine whether the MSC-related profile, along with the biological character of MSC, is maintained by transcription factors expressed selectively in MSC: Oct4, Sox2, c-Myc and Klf4 have been shown to be essential in identifying embryonic stem cells (Takahashi & Yamanaka 2006). And antibodies to MSC-characteristic molecules may be useful in immunohistochemistry, which is important because in vivo distribution status of various stem cells—including hematopoietic stem cells, neuronal stem cells, epidermal stem cells and melanocyte stem cells—is known to be crucial in development and/or tissue repair (Temple 2001; Nishimura et al. 2002; Arai et al. 2004; Fuchs et al. 2004; Benitah et al. 2005).

In this study, we identified MSC-characteristic molecules, including several transcription factors, using human bone marrow MSC in culture: Knockdown of these transcription factors reduced self-renewal capacity and/or differentiation potential. We also found that signature molecule-marked MSC-like cells were present in the bone marrow in vivo.

	Results

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Identification of MSC-characteristic genes

For DNA microarray analyses, we used MSC lines that were not contaminated with fibroblasts (Ishii et al. 2005). The gene expression profile of all examined single cell-derived colonies resembled that of parental MSC population, suggesting there was no developmental heterogeneity (H. Pan and Y. Kato, unpublished data). In addition, we used osteoblasts, chondrocytes and adipocytes that had been differentiated in appropriate differentiation-induction media: Almost all cells in these cultures converted to differentiated cells within 21–28 days (Matsubara et al. 2005). We compared gene expression levels in the MSC (M in Fig. 1, n = 3), osteoblasts (O in Fig. 1, n = 3,), chondrocytes (C in Fig. 1, n = 3) and adipocytes (A in Fig. 1, n = 3), along with fibroblasts (F in Fig. 1, n = 3), using 54 675 DNA microarray probes (Affymetrix). Cluster analysis showed that the gene expression profiles of the three MSC lines, along with one other line, resembled each other but were different from the profiles of all other cells (data not shown), suggesting some small preparation-to-preparation variation within MSC lines. Under these conditions, the expression levels of 148 genes were more than twofold higher in MSC than in any of the differentiated cells and fibroblasts (Table 1). Using real-time RT-PCR (Canales et al. 2006), we also determined that the expression of all 71 examined genes was selectively enhanced in MSC (M, n = 6) compared with various differentiated cells (O, C, or A, n = 6, respectively) and fibroblasts (F, n = 4) (Fig. 1 and data not shown). DNA microarray and real-time RT-PCR analyses showed similar expression patterns of these genes, indicating reproducibility. Figure 1 also shows that LIF as a positive control was expressed at high levels in both MSC and chondrocytes in DNA microarray and real-time RT-PCR analyses.

View larger version (32K): [in this window] [in a new window]   Figure 1  Comparison of DNA microarray and real-time RT-PCR analyses of MSC-characteristic genes. DNA microarray analysis of MSC-characteristic genes was carried out using three lines of each cell type; real-time RT-PCR analysis used six lines each of MSC (M), osteoblasts (O), chondrocytes (C) and adipocytes (A), and four lines of fibroblasts (F). Of 148 MSC-characteristic genes, the differential expressions of 71 genes were confirmed by real-time RT-PCR: This figure includes the expression levels of nine transcription factors, 10 (one overlap) molecules used for immunohistochemistry, and other MSC markers. It also includes profiles of VEGF and LIF mRNA levels: VEGF was included among MSC-characteristic genes based on its profile determined by real-time RT-PCR, and LIF was an MSC/chondrocyte marker (positive control)a. Values represent means ± SEM for three to six cultures. A few values represent means for duplicate cultures.

If some MSC-characteristic genes are indeed associated with the undifferentiated state, their expression should be suppressed upon the onset of differentiation, but in fact most (93%) of these genes, including transcription factor genes, were down-regulated in osteogenic (OP), chondrogenic (CP) and/or adipogenic progenitor cells (AP) within 24 h after incubation with osteogenesis-, chondrogenesis- or adipogenesis-induction medium (Fig. 2A,B and data not shown). During this same 24 h, neither osteoblast-characteristic genes (alkaline phosphatase and PTH receptor), nor chondrocyte-characteristic genes (aggrecan and collagen type II), nor adipocyte-characteristic genes (PPAR and C/EBP) had been up-regulated (Fig. 2C). These findings indicate that most MSC-characteristic molecules are not expressed in committed progenitor cells.

View larger version (27K): [in this window] [in a new window]   Figure 2  Down-regulation of MSC-characteristic genes 24 h after incubation with osteogenic (OP), chondrogenic (CP), or adipogenic induction medium (AP) or medium without differentiation factors (M). (A) The mRNA expression levels were determined by real-time RT-PCR with three MSC lines. (B) Down-regulation of 71 MSC-characteristic genes 24 h after incubation in osteogenesis-, chondrogenesis- or adipogenesis-induction medium. Of the 71 examined genes, the percentages of genes significantly down-regulated 24 h after incubation in one (1 of 3), two (2 of 3) or all (3 of 3) of the three differentiation-induction media are shown. (C) The absence of tissue-specific gene expression at 24 h. MSC were incubated for 24 h (shaded bars) or 28 days (closed bars) with osteogenic (OP), chondrogenic (CP), or adipogenic induction medium (AP) or medium without differentiation factors (M). The mRNA levels were determined by real-time RT-PCR with three MSC lines. The values are means ± SEM for three cultures. M, MSC; OP, osteogenic progenitor cells; CP, chondrogenic progenitor cells; AP, adipogenic progenitor cells; O, osteoblasts; C, chondrocytes; A, adipocytes.

Interestingly, MSC-characteristic genes were found to contain 9 transcription factors—ETV1, ETV5, FOXP1, GATA6, HMGA2, KLF12, PRDM16, SIM2 and SOX11 (Fig. 1)—and most of those except SOX11 were also expressed at high levels in synovial fibroblasts (Table 1). To examine the possible role of these transcription factors, we used siRNA oligonucleotides designed to target these factors: Addition of the siRNA oligonucleotides significantly reduced mRNA levels of most (75%, 53 of 71) examined MSC-characteristic genes within 48–72 h, by which time the levels of the target mRNA for siRNA had decreased by 50–80% (Table 2). Each siRNA down-regulated a different set of MSC-characteristic genes, suggesting that the down-regulation of these genes was not because of any general toxic effects of siRNA. These findings suggest that the 9 transcriptional factors are essential for the molecular signature of MSC.

View larger version (10K): [in this window] [in a new window]   Figure 3  Effects of siRNA for GATA6 on proliferation of bone marrow MSC, synovial fibroblasts and skin fibroblasts. When cultures in 96-well tissue culture plates reached 40% confluency in DMEM supplemented with 10% fetal bovine serum, cells were transfected or not transfected with either an siRNA or a control oligonucleotide in the presence of 10% fetal bovine serum for 24 h and then incubated with DMEM supplemented with 10% fetal bovine serum. Cell numbers were assessed by determination of a ratio of OD at 490 nm to OD at 630 nm using WST-8 reagent. Values represent means ± SEM for three cultures; the variation was small. *P < 0.05, **P < 0.01, ***P < 0.005 compared with negative control siRNA by Student's t-test.

View larger version (24K): [in this window] [in a new window]   Figure 4  Effects of siRNA for MSC-characteristic transcription factors on the increase in alkaline phosphatase activity during osteogenic differentiation of bone marrow MSC. When cultures in 48-well tissue culture plates became 80% confluent, cells were transfected with an siRNA or a control oligonucleotide in the presence of 10% fetal bovine serum for 24 h, and then incubated with DMEM also supplemented with 10% fetal bovine serum for 2 days. These cells were transferred to DMEM supplemented with 10% fetal bovine serum (dot lines) or the osteogenesis-induction medium (solid lines), and incubated for the indicated days. Values represent means ± SEM for three cultures. *P < 0.05, **P < 0.01, ***P < 0.005 compared with negative control siRNA by Student's t-test.

View larger version (21K): [in this window] [in a new window]   Figure 5  Effects of siRNA for MSC-characteristic transcription factors on the increase in GPDH activity during adipogenic differentiation of bone marrow MSC. When cultures in the 48-well tissue culture plates became 80% confluent in DMEM supplemented with 10% fetal bovine serum, cells were transfected with one siRNA or a control oligonucleotide in the presence of 10% fetal bovine serum for 24 h, and then incubated with DMEM supplemented with 10% fetal bovine serum. Two days after the transfection, these cells were transferred to the adipogenesis-induction medium (solid lines) or incubated in DMEM supplemented with 10% fetal bovine serum (dot lines) for the indicated days. Values represent means ± SEM for three cultures. *P < 0.05, **P < 0.01, ***P < 0.005 compared with negative control siRNA by Student's t-test.

We assumed that the identified MSC markers, including non-transcription factors, might be useful in examining immunohistochemistry of the cells. From commercially available antibodies for MSC-characteristic molecules, we selected antibodies against GATA6 (Fig. 1, left panel), ADD3, FLG, HTR7, IGFBP3, MET, TGM2, TRPC4 and VEGF (Fig. 1, right panel), as their mRNA levels were much higher in MSC than in any differentiated cells. We also selected antibodies for LIF, which was expressed at higher levels in MSC and chondrocytes than in osteoblasts, adipocytes, or fibroblasts (Fig. 1, right panel). We conducted a series of dual-immunofluorescent labeling for the MSC population, and found evidence indicating the presence of single- and co-immunopositive cells by confocal fluorescent microscopy: GATA6+ cells (Fig. 6B) and LIF+ cells (Fig. 6C) were present in the interior of bone marrow and near the endosteum of adult mouse humerus, and most of them were GATA6+/LIF+ double positive cells (Fig. 6D,E). Neither a nonspecific serum (Fig. 6F) nor secondary antibodies alone (not shown) showed any specific staining. It is thus unlikely that the doubly stained cells are hematopoietic cells or fibroblasts without multi-potency, as GATA6 and LIF mRNA levels were very low or undetectable in whole bone marrow cells, non-adherent bone marrow cells and skin fibroblasts (Fig. 7).

View larger version (114K): [in this window] [in a new window]   Figure 6  Immunohistochemistry of MSC in adult mouse humerus with anti-GATA6 and anti-LIF antibodies. The bone marrow and bone of 56-day-old humerus were stained with fluorescent secondary antibodies after incubation with anti-GATA6 (B, D and E), anti-LIF antibodies (C, D and E) or a nonspecific serum (F). (A) Hematoxylin–eosin staining of bone marrow and bone. (B) GATA6 was present in the cytoplasm of some bone marrow cells, although it was a transcription factor, which might have been due to the quiescent state of the cells in the tissue. In the bone marrow, comparatively numerous cells were immunopositive for GATA6, and some GATA6-immunopositive cells on the bone surface showed osteoblast-like shapes. GATA6+ cells were also scattered in the periosteum (data not shown). (C) LIF was present in the cytoplasm of some bone marrow cells, and some LIF+ cells were scattered in the outer periosteum (data not shown). (D) The images obtained by confocal fluorescent microscopy and light microscopy were laid so as to overlap each other. (E) Relatively many LIF+/GATA6+ double positive cells were present in the bone marrow and near the endosteum, with a few on the bone surface: The ratio of co-immunopositive cells was high (more than 70%). No double positive cells were detected in the periosteum (data not shown). (F) Negative control with a nonspecific serum. Specific staining was also validated using secondary antibodies alone (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 7  The absence of transcripts for MSC-characteristic genes in non-adherent bone marrow cells. RNA was harvested directly from bone marrow aspirate of a donor (whole BMC), and in addition, bone marrow cells from another donor were seeded and incubated for 24 or 96 h in medium A in plastic tissue culture cells, after which RNA was harvested from non-adherent cells in the medium (24 h Non-adherent BMC or 96 h Non-adherent BMC). RNA was also harvested from the adherent cell layer of MSC when cells were approaching confluence. The mRNA levels of various MSC-characteristic genes in these cells, along with control skin fibroblast lines, were determined by real-time RT-PCR: The mRNA levels in these MSC proved similar to those in other MSC lines. Values represent means of duplicate cultures.

Antibodies for these MSC-characteristic molecules also reacted with some bone marrow cells in vivo. Figure 8 shows a summary of the identification of co-immunopositive cells in mouse bone marrow, using various antibodies. We decided on the combination of antibodies tested for dual immunostaining by taking into account the animal species used for antibody production (rabbit, goat and mouse) and the preservation of immunoreactivity after antigen retrieval by pepsin or microwave treatment. The incidence of co-immunopositive cells was high for TRPC4/FLG (A), medium for LIF/ADD3 (B), GATA6/FLG (C) and TGM2/LIF (D) and low for other combinations (E–N). Among various combinations, GATA6/FLG and TGM2/LIF, along with GATA6/LIF (Fig. 6), could prove useful in identification of MSC-like cells, as these are not expressed in non-adherent bone marrow cells or skin fibroblasts (Fig. 7). TRPC4/FLG may also be useful, as fibroblasts are usually absent in bone marrow (Ishii et al. 2005). The low incidence of single- and co-immunopositive cells with antibodies for MET, IGFBP3 and VEGF may be due to the quality of antibodies and treatment of specimens, or it may be that the mRNA levels of these markers do not always translate to changes in protein levels. Nonetheless, all combinations of antibodies revealed the presence of co-immunopositive cells, which suggests that a fraction of bone marrow cells synthesize several MSC marker proteins simultaneously in vivo.

View larger version (105K): [in this window] [in a new window]   Figure 8  Double staining of MSC-like cells in bone marrow of adult mouse humerus with various combinations of antibodies to MSC-characteristic molecules. MSC-like cells in 56-day-old bone marrow were doubly stained with various antibodies, and then examined using confocal fluorescent microscopy. (A) Comparatively numerous TRPC4+/FLG+ double positive cells were present in the bone marrow and near the endosteum. (B) Comparatively numerous LIF+/ADD3+ cells were present in the bone marrow, and some LIF+/ADD3+ cells were present near the endosteum, but not in the periosteum (data not shown). (C) Many GATA6+/FLG+ cells were present in the bone marrow and some double positive cells were present near the endosteum. No GATA6+/FLG+ cells were detected in the periosteum (data not shown). (D) Comparatively numerous TMG2+/LIF+ cells were present in the bone marrow, and some near the endosteum. (E) Some TRPC4+/IGFBP3+ cells were present in the bone marrow, but none were detected in the periosteum (data not shown). (F) Some TGM2+/MET+ cells were present in the bone marrow and near the endosteum, but no MET+ cells were detected in the periosteum (data not shown). (G) Comparatively numerous LIF+/MET+ cells were detected in the bone marrow, and some LIF+/MET+ cells were present near the endosteum but not in the periosteum (data not shown). (H) One ADD3+/MET+ cell was present near the endosteum, but no ADD3+/MET+ cells were detected in the periosteum (data not shown). (I) A few TRPC4+/VEGF+ cells were present in the bone marrow and near the endosteum but not in the periosteum (data not shown). (J) GATA6+/VEGF+ cells were present near the endosteum, but not on the bone surface, while some GATA6+/VEGF– cells were present on the bone surface. No specific association of VEGF+ or GATA6+ cells with capillaries was observed in the bone marrow. (K) One GATA6+/IGFBP3+ cell was present near the endosteum. (L) The number of GATA6+/MET+ cells was small in the bone marrow. Some double positive cells were present near the endosteum but not on the bone surface. None were detected in the periosteum (Fig. S3 in Supporting Information). (M) The number of FLG+/VEGF+ cells was small, with some near the endosteum. No FLG+/VEGF+ cells were detected in the periosteum (data not shown). (N) The number of FLG+/IGFBP3+ cells in the bone marrow was small: None were detected on the bone surface or in the periosteum (data not shown). (O) Negative control with a nonspecific serum. Specific staining was also validated either with secondary antibodies alone or control IgG (not shown).

In addition, we confirmed that neither chondrocytes (Fig. S2A,C in Supporting Information) nor osteocytes (data not shown) were reactive with antibodies for GATA6, FLG, ADD3 or VEGF (not shown), whereas anti-LIF antibodies did react with chondrocytes (Fig. S2A in Supporting Information), but not with osteocytes (not shown). Therefore, DNA microarray data seems to be relevant to the in vivo situation, at least in part.

	Discussion

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We identified 148 MSC-characteristic genes that were not expressed in any of the differentiated cells or skin fibroblasts, and few of these were expressed in progenitor/precursor cells, even though MSC and skin fibroblasts, along with synovia, were obtained from different donors. A donor-matched comparison will provide more sold data, although it was difficult to obtain donor matched tissues in the present study. We added FGF2 to expand MSC in culture, and this may also have affected the selection of marker genes, even though FGF2 was removed from the cultures 72 h before isolation of RNA. Initial cell density, culture period and passage number could also affect gene expression, so in our study we used the same initial cell density and culture period, and similar passage numbers.

If expanded MSC became heterogeneous due to spontaneous differentiation or maturation, this would also affect the selection of genes. However, our preliminary studies with single cell-derived colonies in low density cultures of MSC showed that all clones have virtually identical MSC marker gene expression profiles, suggesting little developmental hetrogeneity of the MSC.

A comparison of the selected genes among several independent studies may be useful, although culture conditions and microarrays will invariably differ. Wagner selected 25 marker genes that were expressed at higher levels in bone marrow MSC, adipose-derived MSC and cord blood MSC than in fibroblasts (Wagner et al. 2005); Brendel found 21 genes up-regulated in human MSC compared with fibroblasts (Brendel et al. 2005); and Song identified 11 MSC marker genes expressed at higher levels in bone marrow MSC and dedifferentiated MSC-like cells than in osteoblasts, chondrocytes and adipocytes, using DNA microarray (Song et al. 2006). Of the identified genes, FN1, CPNE8, CDH6, NTN4, BDNF, HNT, KIAA0746, NEK7 and UGCG were included in our list, although we have not confirmed that these genes are useful markers. In any case, we found a greater number of MSC-characteristic genes, and confirmed their differential expressions in MSC using real-time RT-PCR.

The present study also revealed that approximately 50% of MSC-characteristic genes are expressed in synovial fibroblasts, which are known to possess higher chondrogenic potential than bone marrow MSC (Sakaguchi et al. 2005): It is known that the occurrence of chondrogenesis in adult physiological bone marrow environment is rear. Nonetheless, it seems significant that both bone marrow MSC and synovial fibroblasts have common (8 of 9) MSC-characteristic transcription factors, although no information as to the possible roles of these transcription factors in MSC is available in the literature. However, EVT1 (Ets variant gene 1) and ETV5 are members of the ETS transcription factor family, some of which induce tumorigenesis of MSC—Ewing sarcoma—when they form a fusion protein with EWS (Riggi et al. 2005). FOXP1 is expressed in various developing tissues in addition to brain, and is required for cardiac growth (Jepsen et al. 2008), and GATA6 is involved in heart/liver/gut development (Morrisey et al. 1996). HMGA2 (HMGI-C)-mutant mice showed the pygmy phenotype (Zhou et al. 1995), and a common variant of HMGA2 is associated with development of adult and childhood height in the general population (Weedon et al. 2007). Furthermore, one SNP of HMGA2 influences adult height, indicating the involvement of HMGA2 in skeletal growth (Weedon et al. 2008). HMGA2 is also expressed in various mesenchymal tissues in embryos, but after birth its expression markedly decreased. However, overexpression of HMGA2 induces mesenchymal tumors, suggesting that HMGA2 stimulates the proliferation of immature mesenchymal cells (Zaidi et al. 2006). PRDM16, a zinc finger transcription factor, is involved in differentiation into brown adipose cells (Seale et al. 2008). SIM2—a basic helix-loop-helix PAS domain (HLH-PAS) transcription factor—is expressed in brain, ribs and limbs, and regulates sonic hedgehog expression (Epstein et al. 2000). SOX11—a Sry-related high-mobility group (HMG) box (Sox) transcription factor—is expressed in various tissues, including skeletogenic primordial (Dy et al. 2008), and SOX11-deficient embryos show skeletal malformations (Sock et al. 2004). In the present study, knockdown of these genes suppressed self-renewal of MSC, so all these results, taken together suggest the involvement of these transcription factors in stem cell expansion during development.

Unexpectedly, the siRNA for MSC-characteristic transcription factors had a modest or limited effect on the differentiation potential, so combinations of siRNA for several factors may be required to abolish the differentiation potential. More extensive studies on the individual assigned transcription factor and/or its mutual interactions in various aspects are required for the comprehensive understanding of MSC multi-potency.

Another significant finding in this study was identification of signature molecule-marked MSC in vivo: Observation of doubly immunostained cells using confocal fluorescent microscopy helped us to distinguish single- and co-immunostained cells in mouse bone marrow in a dim background. Interestingly, all combinations of the antibodies that could be tested proved the presence of co-immunopositive cells in mouse bone marrow, and none of these combinations supported the presence of co-immunopositive cells in the perichondrium/periosteum, although singly immunopositive cells were detected. The antibody set was not useful in identification of perichondrium/periosteum MSC, probably because the MSC markers were selected using bone marrow MSC lines. The protein expression profile of perichondrium/periosteum MSC may differ somewhat from that of bone marrow MSC.

As GATA6 and LIF, along with FLG, TGM2 and HTR7, were not expressed in non-adherent bone marrow hematopoietic cells or fibroblasts, the adherent bone marrow cells concurrently expressing these molecules are likely to be multi-potent MSC. Although LIF is not a marker for MSC alone, anti-LIF antibodies appear to be useful in examining the immunohistochemistry of MSC in the absence of cartilage.

We could not detect a specific association of co-immunopositive cells with blood vessels, although a relation between MSC and pericytes has been suggested (Traktuev et al. 2008).

MSC-like cells with multi-potency have recently been isolated from virtually all postnatal organs and tissues, including adipose tissue, muscle, lung and pancreas (Zuk et al. 2001; Mastrogiacomo et al. 2005; Sakaguchi et al. 2005; da Silva Meirelles et al. 2006). Some of these cells may have reverted from the differentiation stage to the stem cell stage under culture conditions (Sakaguchi et al. 2005; Song et al. 2006), but in preliminary studies we could not detect MSC-like cells in these non-skeletal tissues using the antibody set, probably because these cells show different protein expression profiles in vivo.

In this study, we used mouse tissue samples in immunohistochemistry, but it must be noted that the properties of human and mouse bone marrow MSC may be different. Thus, the immunohistochemistry of human MSC may provide more information in the future.

In conclusion, we found that the molecular signature of bone marrow MSC is determined by transcription factors that are expressed selectively in these cells. And some of these transcription factors are apparently involved in MSC stemness regulation, and may provide tools for studies on the molecular basis of the stemness. Furthermore, we identified MSC-like cells in vivo: The location of MSC-like cells in bone marrow suggests that the stem cells are physiologically involved in bone development/growth, and adult bone marrow proved to be a good source of native MSC. These findings should promote the use of adult bone marrow MSC in regenerative medicine, and the antibodies for some MSC-characteristic molecules may be useful in characterization of "MSC niches" and studies on the roles of MSC in various physiological and pathological situations in vivo.

	Experimental procedures

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Cell cultures

Human iliac MSC were purchased from BioWhittaker Inc (Walkersville, MD) or obtained from iliac crest according to protocol approved by ethical authorities at Hiroshima University. Human skin fibroblasts were purchased from Kurabo (Osaka, Japan), and human gingival fibroblasts were isolated as described previously (Kawahara & Shimazu 2003). Human osteoarthritic synovial fibroblasts (MSC-like cells) were purchased from Cell Applications Inc (San Diego, CA). Cultures of MSC and their differentiation from the osteoblast, chondrocyte or adipocyte lineage were carried out as previously described (Pittenger et al. 1999; Matsubara et al. 2005): We used FGF-2 at 1 ng/mL to expand MSC because MSC expanded ex vivo with FGF-2 maintained their multi-differentiation potential throughout many mitotic divisions (Tsutsumi et al. 2001). To avoid direct action of FGF-2 on gene expression, FGF-2 was removed from the culture medium of MSC or fibroblasts 72 h before isolation of RNA.

DNA microarray analysis

Total RNA was isolated from confluent cultures of three lines of MSC (passage 6–9), three lines of fibroblasts (passage 7–14), three lines of MSC incubated for 24 h or 28 days in the differentiation-induction medium, or three lines of osteoarthritic synovium-derived MSC (passage 6–8), using RNeasy Mini Kit (Qiagen, Chatsworth, CA). DNA microarray analysis was carried out by KURABO GeneChip Custom Analysis Service with Human Genome U133 Plus 2.0 chips containing 38 500 genes/47 000 transcripts variants/54 000 probes (Affymetrix. Inc., Santa Clara, CA). The raw data (Microarray Suite version 5.0, SF = 1; Affymetrix Inc.) were standardized by the global median normalization method using GeneSpring (Silicon Genetics, Redwood City, CA). Normalization was limited by flag values, and the median was calculated using genes that exceeded the Present or Marginal flag restriction. Gene filtering on normalized intensity followed by fold changes (more than twofold) was used to generate the list of genes for expression profiles before or after differentiation. The raw data were deposited in the GEO (GSE9451 [NCBI GEO] ).

Real-time RT-PCR

Real time quantitative RT-PCR analyses were carried out using ABI Prism 7900HT Sequence Detection System instrument and software (Applied Biosystems, Inc., Foster City, CA). The primers and probes were purchased from Applied Biosystems, Inc. Data were normalized against 18S rRNA levels.

Effects of siRNA for MSC-characteristic transcription factors on the expression of numerous MSC-characteristic genes

siRNA oligonucleotides designed to target the nucleotide sequence for ETV1, ETV5, FOXP1, GATA6, HMGA2, KLF12, PRDM16, SIM2 and SOX11, along with a control oligonucleotide, were obtained from RNAi Co., Ltd. (Tokyo, Japan). The siRNA oligonucleotides were transfected into MSC on 12-well plates (22-mm diameter) in the presence of 10% fetal bovine serum using Lipofectamin 2000 (Invitrogen, Carlsbad, CA) following the procedure of the manufacturer. Efficiency of knockdown 48 h (n = 2) and 72 h (n = 2) after transfection was examined by real-time RT-PCR.

Cell proliferation, alkaline phosphatase, GPDH, alizarin red staining and oil red O staining

When MSC or synovial fibroblast or skin fibroblast cultures in 24-well tissue culture plates became 40% confluent, cells were not transfected or transfected with each siRNA or a control oligonucleotide, using Lipofectamin 2000 as described above. The cells were transferred to Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum 24 h after transfection, and incubated for more 2–6 days. Cell number was assessed using WST-8 reagent (Seikagaku Co., Tokyo, Japan). To examine the effect of knockdown on the differentiation potential, cells in 48-well multi-well tissue culture plate were transfected with siRNA or a control oligonucleotide using Lipofectamin 2000 as described above, when the cultures became 80% confluent. The cultures were transferred to the osteogenesis or adipogenesis-induction medium 2 days after transfection. The alkaline phosphatase activity was determined be the method of Bessey et al. (1946). The GPDH activity was determined using a GPDH assay kit (Hokudo, Sapporo, Japan) (Guo et al. 2000).

Immunohistochemistry

C57/BL/6J mice (0.5 and 56-day-old) and pregnant mice of embryonic day 15.5 were used. The animals were anesthetized using sodium pentobarbital and transcardially perfused with 4% paraformaldehyde, after which multiple organs and tissues were dissected. Limb specimens were decalcified with 10% EDTA at 4 °C. The specimens were paraffin-embedded, and serial sections (4 µm thick) were prepared with a rotary microtome (HM355, MICROM, Germany). Antibodies used were polyclonal goat anti-human FLG (1 : 100, sc-25896, Santa Cruz Biotech., USA), monoclonal mouse anti-human IGFBP3 (1 : 500, I4527, Sigma), monoclonal mouse anti-human VEGF (1 : 1000, 05443, Upstate Inc., USA), polyclonal rabbit anti-human GATA6 (1 : 500, sc-9055, Santa Cruz Biotech), polyclonal rabbit anti-mouse TRPC4 (1 : 2000, ACC018, Alomone Labs Ltd., Israel), polyclonal rabbit anti-human TGM2 (1 : 10, AB15536, Abcam Ltd., USA), polyclonal rabbit anti-human HTR7 (1 : 300, AB5661, Chemicon Int. Inc., USA), polyclonal rabbit anti-human ADD3 (1 : 500, sc-25733, Santa Cruz Biotech), monoclonal mouse anti-mouse MET (1 : 500, sc-8057, Santa Cruz Biotech), and polyclonal goat anti-human LIF (1 : 500, sc-1336, Santa Cruz Biotech). Antigen retrieval was conducted either by 0.1% pepsin treatment for 10 min at 37 °C with 10 mM HCl—in the cases of VEGF, GATA6, TRPC4, HTR7, IGFBP3 and FLG—or by microwave (H2800, Energy Beam Sciences, Inc., USA) treatment in 10 mM citrate buffer, pH 6.0 for 10 min at 90 °C, in the cases of TGM2, MET, ADD3 and LIF. Immunoreactivity was visualized using a combination of Alexa Fluor 567 (excited at 568 nm, red) and Alexa Fluor 647 (excited at 647 nm, green). Cell nuclei were stained with DAPI (blue, S24535, Molecular Probes, USA). After recording confocal fluorescent images, the same specimens were stained with hematoxylin–eosin (HE) and then the localization of co-immunopositive cells was validated by overlapping of the corresponding immunofluorescence and HE-stained cellular images. The incidence of doubly immunostained cells was estimated by counting co-immunopositive cells and single positive cells in three to four pictures. We categorized the incidence of co-immuopositive cells among all positive ones into three grades: high (> 70%), medium (30–70%), and low (< 30%). In some studies, immunoreactivity was visualized with 3,3'-diaminobenzidine (DAB; SK-4100, Funakoshi, Japan) coloring by the ABC method (ABC Elite kit, Vector, USA).

	Acknowledgements

 
This study was supported by a fund from the Japan Science and Technology Agency (JST), Japan and Health and Labour Sciences Research Grants on Tissue Engineering (H17-022) from the Japanese Ministry of Health, Labour and Welfare.

	Footnotes

 
Communicated by: Yoshiaki Ito

H. Kubo and M. Shimizu contributed equally to this article.

^* Correspondence: ykato{at}hiroshima-u.ac.jp

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Received: 19 June 2008
Accepted: 6 December 2008

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