Combined Omics Analysis Identifies Transmembrane 4 L6 Family Member 1 as a Surface Protein Marker Specific to Human Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) are promising for cell therapy and regenerative medicine; but their lack of specific markers renders the cell culture at potential contamination risk with other cell types, in particular, fibroblasts. In this study, we mapped 2 differential transcriptome data of MSCs compared, one to mononuclear cells and the other to fibroblasts, onto the membrane proteome data, the analysis of which led to an identification of transmembrane 4 L6 family member 1 (TM4SF1) as a surface protein marker candidate that could discriminate MSCs simultaneously from blood cells and fibroblasts. Our analyses confirmed that TM4SF1 was abundantly expressed on MSCs but neither on other blood/tissue cells nor on fibroblasts. TM4SF1 immunoselection from bone marrow and adipose tissues yielded homogeneous cell populations that were highly similar to MSCs, in terms of morphology, immunophenotype, and differentiation potential. These findings indicate that TM4SF1 can serve as a surface protein marker which singly identifies MSCs from diverse cell sources, in particular, fibroblast-rich connective tissues.

Introduction

A pool of mesenchymal stem cells (MSCs) or mesenchymal stromal cells serves not only as a cell reservoir for regeneration of connective tissues [1] but also as a trophic regulator of hematopoiesis [2] and immune responses [3]. This multifaceted functionality of MSCs confers diverse therapeutic potential, the translation of which into clinically relevant applications, however, has been limited by the concern over the heterogeneity [4 –6] and/or potential contamination of MSC cultures with other types of blood and tissue cells. In particular, fibroblasts are the contaminants of primary concern, because they are not only abundant and omnipresent in the body but also share extensive phenotypic and functional similarities with MSCs [7]. Therefore, fibroblasts are likely to contaminate MSC cultures during procurement of tissue sources; and those contaminants, once introduced to MSC harvests, are hardly distinguishable from MSCs and, thus, remain as an inseparable entity until the end of mass production.

Over the past decades, advances in hybridoma technology have expedited the development of a series of MSC-reactive monoclonal antibodies, namely SH-2, SH-3, SH-4, SB-10, HOP-26, and Stro-1 [8 –12], which have turned later out to recognize several different surface proteins on MSCs, including CD105, CD73, CD166, and CD63 [13 –15]. Recently, 3 glycolipids, stage specific embryonic antigen (SSEA)-1 [16], SSEA-4 [17], and disialoganglioside (GD2) [18] as well as 3 transmembrane proteins, nerve growth factor receptor [19], CD200 [20], and fibroblast activation protein α (FAPα) [21] have been discovered as MSC-specific surface markers and proved effective in identification and enrichment of MSCs from bone marrow (BM). However, it remains largely unknown whether all these molecules can equally serve as MSC-specific markers in other tissue sources and, more importantly, whether they can reliably discriminate MSCs from their phenotypic twin cells, that is, fibroblasts.

Tetraspanins are a large family of membrane glycoproteins that display a characteristic structural feature made of 4 transmembrane domains. There is a separate group of tetraspanins, called a transmembrane 4 L6 family (TM4SF), that share limited sequence homology with other family members [22]. They are known to induce formation of membrane microdomains and play diverse roles in various blood cells (for review, see ref. [23]); but little is known about their expression and roles in MSCs, except the role of CD9 in cell proliferation and attachment [24].

In this study, we performed a combined analysis on previously obtained MSCs' membrane proteome data [25] and differential transcriptome data [26,27], in an attempt to discover novel surface protein markers that could simultaneously distinguish MSCs from blood cells and fibroblasts. Such surface markers would facilitate identification and enrichment of MSCs from diverse cell sources, in particular, fibroblast-rich connective tissues.

Materials and Methods

Cell preparation

Cryopreserved human BM-derived mononuclear cells (MNCs), BM-derived MSCs, adipose tissue (AT)-derived MSCs, and dermal fibroblasts were purchased from Lonza Inc. Cryopreserved AT-derived stromal vascular fraction (SVF) samples as well as umbilical cord blood (UCB)-derived MNCs and MSCs were obtained from local companies with informed written consents. Both MSCs and fibroblasts were cultivated as previously reported [27]. A hybridoma cell line (L6-20-4) producing anti-TM4SF1 antibody was a generous gift from Prof. Steve R. Roffler (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan).

Flow cytometry

All analyses were performed on a Guava PCA-96 flow cytometer (Guava Technlogies), with mouse monoclonal antibodies against previously established MSC markers (Table 1) and Alexa532-conjugated goat anti-mouse IgG (Invitrogen). All measurements were analyzed with Guava Express software and De novo software.

Table 1.

List of Antibodies Used in the Flow Cytometry Analysis

Antibody	Target	Isotype	Vendor
SH2	CD105 (endoglin)	IgG1	Santa Cruz Biotechnology, Inc.
SH3/SH4	CD73 (NT5E)	IgG3	Santa Cruz Biotechnology, Inc.
SB-10	CD166 (ALCAM)	IgG1	Millipore Co.
HOP-26	CD63 (LAMP-3)	IgG1	Millipore Co.
Anti-SSEA-4	Globo-series glycolipids	IgG3	Millipore Co.
Anti-GD2	Disialoganglioside	IgG2a	Millipore Co.
Anti-CD49a	CD49a (integrin α)	IgG1	Millipore Co.
Anti-CD146	CD146 (MCAM)	IgG1	Millipore Co.
Anti-FAPα	FAPα	IgG1	Santa Cruz Biotechnology, Inc.
Anti-CD90	CD90 (Thy-1)	IgG2a	Santa Cruz Biotechnology, Inc.

Reverse transcriptase-polymerase chain reaction analysis

Human multiple tissue cDNA panels were obtained from Clonetech Laboratories Inc. For other cell samples, total RNA was extracted using Trizol Reagent and reverse-transcribed to cDNA, as previously reported [25]. Primer sets and amplification conditions for the genes were listed in Table 2.

Table 2.

Primers and Conditions Used for the Reverse Transcriptase–Polymerase Chain Reaction Analysis

Gene	Sequence	Product size (bp)	Anneal temperature (°C)
TM4SF1	F 5′-CCTAGCAGACCACCA − 3′ R 5′-GTCCTGGGTTGGTTCT-3′	640	57
BSP	F 5′-GCACGCCTACTTCTATCCTC-3′ R 5′-CGGCCTCGG AGTCTT-3′	185	59
RUNX2	F 5′-ACTGGGCCCTTTTTCAGA-3′ R 5′-GCGGAAGCATTCTGGAA-3′	317	54
aP2	F 5′-GGCCAAACCCAACCTGA-3′ R 5′-GGGCGCCTCCATCTAAG-3′	167	57
PPAR-γ2	F 5′-GCTGTTATGGGTGAAACTCTG-3′ R 5′-ATAAGGTGGAGATGCAGGTTC-3′	350	62
COLX	F 5′-GCCCAAGAGGTGCCCCTGGAATAC-3′ R 5′-CCTGAGAAAGAGGAGTGGACATAC-3′	703	57
SOX9	F 5′-AGACAGCCCCCTATCGACTTC-3′ R 5′-TGCTGCTTGGACATCCACAC-3′	230	63
ACTB	F 5′-AGAAAATCTGGCACCACACC-3′ R 5′-CCATCTCTTGCTCGAAGTCC-3′	435	57

BSP, bone sialoprotein; RUNX2, runt-related transcription factor 2; aP2, adipocyte fatty acid binding protein; PPAR-γ2, peroxisome proliferator-activated receptor γ2; COLX, collagen X; SOX9, Sry-box 9; ACTB, β-actin.

Omics data manipulation

The membrane proteome data of MSCs [25] consisting of 191 ectodomain-containing membrane-bound proteins were integrated with 2 differential transcriptome datasets of MSCs [26,27] and reduced into a subset of 148 different proteins (Supplemental Table S1, available online at www.liebertonline.com/scd) by eliminating proteins whose gene expression values were absent in either of the 2 transcriptome datasets.

Fluorescence cell labeling

For efficient fluorescence labeling and tracing of MSCs, we used Cell Staker (Biterials Co., www.biterials.com), nontoxic, and non-photobleachable nanoparticles with the size of 50 nm containing rhodamine B isothiocyante in the silica shell [28]. Two milligram of nanoparticles were washed thrice with PBS and mixed with 10 ml of culture media. After about 3 × 10⁵ attached MSCs were incubated overnight with nanoparticle-containing media, they were washed twice with PBS, trypsinized, and measured by flow cytometry.

Immunoselection

About 0.5 μg of L6-20-4 monoclonal antibodies purified from the supernatant of hybridoma cultures were incubated with 25 mL (1 × 10⁷) of Dynabeads Pan mouse IgG (Dynal Biotech) for 30 min. After removal of excess antibody, the coated beads were then mixed with 2.5 × 10⁷ thawed BM-derived MNCs or AT-derived SVFs, incubated for 20 min at 4°C, and separated by applying external magnets.

In vitro differentiation assays

TM4SF1⁺ cells were in vitro expanded to a second passage and incubated with mesenchymal differentiation media (Lonza, Inc.). Their differentiation into osteogenic, adipogenic, and chondrogenic lineages were measured by von Kossa, Oil Red O, and Alcian Blue staining, respectively.

In vivo osteogenic differentiation assay

The 0.4 μm-pore size transwell inserts (Corning, Inc.) were seeded with about 1 × 10⁴ cells, sealed with parafilm, and subcutaneously implanted for 3 weeks in nude mice. After the inserts were harvested, their membranes were detached and stained by von Kossa method.

Results and Discussion

We first compared the expression pattern between MSCs and fibroblasts using previously established MSC markers (Table 1). The flow cytometry analysis showed that MSCs were strongly positive for CD105, CD73, CD166, CD63, SSEA-4, GD2, and FAPα and weakly positive for CD49a and CD146 (Fig. 1A), thereby certifying individual or combinatorial uses of these surface markers for positive identification of MSCs. Unfortunately, however, most of these markers were found to be expressed also on skin fibroblasts, mostly at comparable levels to those on MSCs, indicating that these markers might be unsuitable for discriminating unequivocally between MSCs and fibroblasts. Exceptionally, GD2 appeared to be differentially expressed in MSCs compared with fibroblasts, implying its potential use to discriminate MSCs from fibroblasts. However, its highly dispersed and heterogeneous expression pattern, which was also shown in a previous report [18], would limit the widespread use in basic and clinical research.

FIG. 1.

Identification of TM4SF1 as a novel MSC-specific surface marker. (A) Comparison in the surface phenotype between MSCs (shaded profiles) and fibroblasts (open profiles) with a panel of previously established MSC markers. Both cell populations were expanded to the fifth passage, incubated with mouse monoclonal antibodies against indicated markers and Alexa532-conjugated secondary antibody. The control profiles were obtained from the cells stained only with the secondary antibody. Note that all MSC markers, except GD2, were cross-reactive to fibroblasts. (B) The 2D scatter plot analysis of the combined omics data in which a membrane proteome was integrated with 2 differential transcriptome of MSCs, 1 against MNCs and the other against fibroblasts. Each black dot indicates a transmembrane protein whose gene expression ratios in MSCs compared with fibroblasts and MNCs are presented in a logarithmic scale by X and Y coordinates, respectively. Note that there was a single protein (TM4SF1) whose coordinates are greater than 10 in both dimensions. (C) RT-PCR analysis showing preferential TM4SF1 expression in BM-derived MSCs over MNCs and fibroblasts. (D) Comparative flow cytometry analysis demonstrating that TM4SF1 was strongly expressed on BM-derived MSCs (a heavily shaded profile) but not on MNCs (a lightly shaded profile) and fibroblasts (an open profile). (E) RT-PCR analysis demonstrating that MSCs derived from umbilical cord blood and adipose tissue could express TM4SF1 at comparable levels to that in BM-derived MSCs (F, G) multiple tissue cDNA panel screening showing null or negligible TM4SF1 expression in human blood fractions and in major human organ tissues. Although lung and pancreas tissues were observed to express TM4SF1 at detectable levels, in accordance to antibody-based proteome profiling data (www.proteinatlas.org), their expression levels were much lower compared with those in MSCs. BM, bone marrow; MSC, mesenchymal stem cell; MNC, mononuclear cell; RT-PCR, polymerase chain reaction.

Therefore, of practical value for safe and effective clinical applications of MSCs would be novel surface markers that could discriminate reliably MSCs not only from other blood cells but also from fibroblasts. To discover such doubly discriminating surface markers, we devised a combined omics approach, in which the membrane proteome data of MSCs [25] were integrated with 2 differential transcriptome datasets of MSCs, 1 against BM-derived MNCs [26] and the other against fibroblasts [27], and reduced to a subset of membrane proteins whose gene expression intensity ratios in MSCs compared with MNCs and fibroblasts could be mapped (Supplemental Table S1). A 2D scatter plot analysis of the combined data revealed TM4SF1, variously known as H-L6, L6, M3S1, or TAAL6, as a leading surface marker candidate that could simultaneously distinguish MSCs from MNCs and fibroblasts, because its gene expression was observed to be uniquely up-regulated at 10-fold or higher levels in MSCs compared with the other 2 cell populations (Fig. 1B).

To verify this finding, we compared TM4SF1 expression among BM-derived MSCs, MNCs, and fibroblasts at both gene and protein levels. The reverse transcriptase–polymerase chain reaction (PCR) followed by flow cytometry analyses showed that TM4SF1 was abundantly expressed on MSCs but neither on fibroblasts nor on MNCs (Fig. 1C, D), indicating that TM4SF1 expression might be exclusive to MSCs within BM stroma. Likewise, TM4SF1 expression also appeared to be highly confined to a MSC type within 2 other important sources for MSCs, that is, UCB and AT, because a high level TM4SF1 expression was observed in MSCs derived from UCB and AT but not in those tissues per se (Fig. 1E) where MSCs were very rare in number.

Then, we examined TM4SF1 expression in major body tissues by PCR screening of human multiple tissue cDNA panels. The results showed that TM4SF1 was not expressed at any significant level in either peripheral blood fractions (Fig. 1F) or major tissues (Fig. 1G). These findings were in line with previous reports that TM4SF1 expression, albeit often up-regulated in some carcinomas [29], was suppressed to a very low level in most of normal tissues [30]. Therefore, TM4SF1 is likely to serve as a surface protein marker that can uniquely identify MSCs from most of tissue sources, including fibroblast-rich connective tissues.

To prove such a possibility, we attempted to isolate MSCs from cryopreserved BM samples by immunoselection with TM4SF1-targeting antibodies. For this study, we used an L6-20-4 antibody [31], whose high specificity and affinity toward the ectodomain of TM4SF1 was demonstrated in our feasibility test with a cell mixture of TM4SF1⁺ MSCs and TM4SF1⁻ fibroblasts (Supplementary Fig. S1, available online at www.liebertonline.com/scd). When TM4SF1-based immunoselection was performed on thawed BM samples, adherent TM4SF1⁺ cells could be isolated with a frequency of about 1 in 5 × 10⁶ cryopreserved MNCs. Each TM4SF1⁺ cell, on isolation, grew rapidly to form a colony of homogeneous fibroblastic cells (Fig. 2A). The TM4SF1⁻ fraction, on the other hand, yielded no such colony of fibroblastic cells, suggesting that TM4SF1 might be expressed on only a very few number of BM cells, all of which, nonetheless, could assume an adherent fibroblastic phenotype.

FIG. 2.

Comparative analyses of morphology, immunophenotype, and mesengenic differentiation potential between TM4SF1⁺ and TM4SF1⁻ cells (A) A photomicrograph of a representative cell colony of TM4SF1⁺ cells isolated from cryopreserved BM-derived MNCs (magnification 40 × ). Images of (B) TM4SF1⁺ and (C) TM4SF1⁻ adherent cells immunoselected from adipose tissue-derived stromal vascular fraction cells. (D) Flow cytometric analysis demonstrating an immunophenotypic similarity between TM4SF1⁺ (a close profile) and TM4SF1⁻ cells (an open profile). The cells stained only with the secondary antibody were used as the negative control. (E–J) Comparative cytochemical staining and RT-PCR analyses of in vitro differentiated cells. Both TM4SF1⁺ and TM4SF1⁻ cells derived from stromal vascular fraction were culture expanded and separately incubated under (E, F) osteogenic, (G, H) adipogenic, and (I, J) chondrogenic conditions and assessed for their differentiation potential by (E) Von Kossa, (G) Oil Red O, and (I) Alcian Blue stains, respectively, as well as by RT-PCR analyses of (F) bone-related BSP and RUNX2 genes, (H) fat-related aP2 and PPAR-γ2 genes, and (J) cartilage-related COLX and SOX9 genes, respectively. (K) In vivo osteogenic differentiation assay. Cells were loaded on the transwell inserts, sealed, and subcutaneously implanted in nude mouse. After 3 weeks, the membranes of the inserts were stained by von Kossa method. Color images available online at www.liebertonline.com/scd.

Next, to demonstrate that TM4SF1 could serve as a surface marker discriminating MSCs simultaneously from fibroblasts and MNCs, we repeated immunoselection with a cryopreserved SVF sample, one of fibroblast-rich cell sources [32]. The results showed that both TM4SF1⁺ and TM4SF1⁻ fractions yielded fibroblastic cells with frequencies of about 1 to 10 in 1 × 10⁴ cryopreserved SVF cells. On continuous cultivation, TM4SF1⁺ cells yielded a homogenous fibroblastic cell population (Fig. 2B), whereas the TM4SF1⁻ cells gave rise to heterogeneous cell types (Fig. 2C). Although TM4SF1⁻ cells exhibited a slightly higher growth rate than TM4SF1⁺ cells (Supplementary Fig. S2, available online at www.liebertonline.com/scd), both cell populations could continue to grow over 12–13 passages, without showing any signs of senescence and transformation. Intriguingly, both TM4SF1⁺ and TM4SF1⁻ cells shared similar surface expression patterns for CD63, CD73, CD90, CD105, and CD166 (Fig. 2D), all of which were known as established diagnostic markers for MSCs.

To further investigate the nature of TM4SF1⁺ and TM4SF1⁻ cells, we incubated them separately under mesengenic differentiation conditions. Cytochemical staining and PCR analyses showed that TM4SF1⁺ cells, but not TM4SF1⁻ cells, could readily differentiate into osteoblasts, adipoblasts, and chondroblasts (Fig. 2E–J; for MSC control, Supplementary Fig. S3, available online at www.liebertonline.com/scd). Further, an in vivo ectopic bone formation assay also showed that TM4SF1⁺ cells could be induced to differentiate along an osteogenic lineage (Fig. 2K). Given the close immunophenotypic similarity, these notable differences in differentiation potential between the 2 cell populations were highly reminiscent of those observed between MSCs and fibroblasts [27], implying that TM4SF1⁺ cells would represent a homogeneous MSC population; whereas adherent TM4SF1⁻ cells were heterogeneous fibroblasts. Therefore, it is very likely that TM4SF1 can serve as a surface protein marker for selection of MSCs, not only from fibroblast-free BM stroma but also from fibroblast-rich fat tissues.

In the future, TM4SF1, due to its doubly discriminative nature against fibroblasts and blood cells, would be popularly used for identification and enrichment processes of MSCs from diverse cell sources, in particular, fibroblast-rich connective tissues.

Footnotes

Acknowledgments

This research was supported in part by a Korea Science and Engineering Foundation grant (M10641000037) funded by the Ministry of Education, Science, and Technology and in part by the grant (SC-2140) from Stem Cell Research Center of the 21st Century Frontier Research Program, Republic of Korea.

Author Disclosure Statement

No competing financial interests exist.

References

Pittenger

, Mackay

, Beck

, Jaiswal

, Douglas

, Mosca

, Moorman

, Simonetti

, Craig

, Marshak

. 1999. Multilineage potential of adult human mesenchymal stem cells. Science, 284:143–147.

Dazzi

, Ramasamy

, Glennie

, Jones

, Roberts

. 2006. The role of mesenchymal stem cells in haemopoiesis. Blood Rev, 20:161–171.

Uceelli

, Moretta

, Pistoia

. 2006. Immunoregulatory function of mesenchymal stem cells. Eur J Immunol, 36:2566–2573.

Addallah

, Kassem

. 2008. Human mesenchymal stem cells: from basic biology to clinical applications. Gene Ther, 15:109–116.

Wagner

, Ho

. 2007. Mesenchymal stem cell preparations-comparing apples and oranges. Stem Cell Rev, 3:239–248.

Jones

, McGonagle

. 2008. Human bone marrow mesenchymal stem cells in vivo. Rheumatology, 47:126–131.

Lysy

, Smets

, Sibille

, Najimi

, Sokal

. 2007. Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation. Hepatol, 46:1574–1585.

Haynesworth

, Barber

, Caplan

. 1992. Cell surface antigens on human marrow-derived mesenchymal cells are detected by monoclonal antibodies. Bone, 13:69–80.

Bruder

, Horowitz

, Mosca

, Haynesworth

. 1997. Monoclonal antibodies reactive with human osteogenic cell surface antigens. Bone, 21:225–235.

10.

Joyner

, Bennett

, Triffitt

. 1997. Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies. Bone, 21:1–6.

11.

Simmons

, Torok-Storb

. 1991. Identification of stromal cell precursors in human bone marrow by a novel monoclonal antibody, STRO-1. Blood, 78:55–62.

12.

Barry

, Boynton

, Haynesworth

, Murphy

, Zaia

. 1999. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an epitope on endoglin (CD105) Biochem Biophys Res Comm, 265:134–139.

13.

Barry

, Boynton

, Murphy

, Zaia

. 2001. The SH-3 and SH-4 antibodies recognize distinct epitopes on CD73 from human mesenchymal stem cells. Biochem Biophys Res Comm, 289:519–524.

14.

Bruder

, Ricalton

, Boynton

, Connolly

, Jaiswal

, Zaia

, Barry

. 1998. Mesenchymal stem cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation. J Bone Miner Res, 13:655–663.

15.

Zannettino

ACW

, Harrison

, Joyner

, Triffitt

, Simmons

. 2003. Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody, HOP-26. J Cell Biochem, 89:56–66.

16.

Anjos-Afonso

, Bonnet

. 2007. Nonhematopoietic/endothelial SSEA-1+ cells define the most primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood, 109:1298–1306.

17.

Gang

, Bosnakovski

, Figueiredo

, Visser

, Perlingeiro

RCR

. 2007. SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood, 109:1743–1751.

18.

Martinez

, Hofmann

, Marino

, Dominici

, Horwitz

. 2007. Human bone marrow mesenchymal stromal cells express the neural ganglioside GD2: a novel surface marker for the identification of MSCs. Blood, 109:4245–4248.

19.

Quirici

, Soligo

, Bossolasco

, Servida

, Lumini

, Deliliers

. 2002. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol, 30:783–791.

20.

Delorme

, Ringe

, Gallay

, Le Vern

, Kerboeuf

, Jorgensen

, Rosset

, Sensebe

, Layrolle

, Haupl

, Charbord

. 2008. Specific plasma membrane protein phenotype of culture-amplified and native human bone marrow mesenchymal stem cells. Blood, 111:2631–2635.

21.

Bae

, Park

, Son

, Ju

, Paik

, Jeon

, Koh

, Kim

. 2008. Fibroblast activation protein alpha identifies mesenchymal stromal cells from human bone marrow. Brit J Haematol, 142:827–830.

22.

Wright

, Ni

, Rudy

. 2000. The L6 membrane proteins - a new four-transmembrane superfamily. Protein Sci, 9:1594–1600.

23.

Tarrant

, Robb

, van Spriel

, Wright

. 2003. Tetraspanins: molecular organizers of the leukocyte surface. Trends Immunol, 24:610–617.

24.

Kim

, Yu

, Joo

, Kim

, Cho

, Bae

, Jung

. 2007. Role of CD9 in proliferation and proangiogenic action of human adipose-derived mesenchymal stem cells. Pflugers Arch, 455:283–296.

25.

Jeong

, Ko

, Park

, Lee

, Jang

, Jeon

, Koh

, Kim

. 2007. Membrane proteomic analysis of human mesenchymal stromal cells during adipogenesis. Proteomics, 7:4181–4191.

26.

Jeong

, Ko

, Bae

, Jeon

, Koh

, Kim

. 2007. Genome-wide differential gene expression profiling of human bone marrow stromal cells. Stem Cells, 25:994–1002.

27.

Bae

, Ahn

, Park

, Son

, Kim

, Lim

, Jeon

, Kim

. 2008. Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts. Cell Tissue Res, 335:565–573.

28.

Park

, Tae

, Choi

, Kim

, Moon

, Kim

, Lee

, Kim

, Park

, Lee

, Joh

, Kim

. 2010. Characterization, in vitro cytotoxicity assessment, and in vivo visualization of multimodal, RITC-labeled, silica-coated magnetic nanoparticles for labeling human cord blood-derived mesenchymal stem cells. Nanomedicine, 6:263–276.

29.

Marken

, Schieven

, Hellstrom

, Aruffo

. 1992. Cloning and expression of the tumor-associated antigen L6. Proc Natl Acad Sci USA, 89:3503–3507.

30.

Marken

, Bajorath

, Edwards

, Farr

, Schieven

, Hellstrom

, Aruffo

. 1994. Membrane topology of the L6 antigen and identification of the protein epitope recognized by the L6 monoclonal antibody. J Biol Chem, 269:7397–7401.

31.

Hellstrom

, Horn

, Linsley

, Brown

, Brankovan

, Hellstrom

. 1986. Monoclonal mouse antibodies raised against human lung carcinoma. Cancer Res, 46:3917–3923.

32.

Yoshimura

, Shigeura

, Matsumoto

, Sato

, Takaki

, Aiba-Kojima

, Sato

, Inoue

, Nagase

, Koshima

, Gonda

. 2006. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol, 208:64–76.

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