Biological Activities Encoded by the Murine Mesenchymal Stem Cell Transcriptome - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:13 |只看该作者 |倒序浏览 |打印

作者：Donald G. Phinney, Katy Hill, Charles Michelson, Maria DuTreil, Catherine Hughes, Sally Humphries, Robin Wilkinson, Melody Baddoo, Erica Bayly作者单位：Center for Gene Therapy, Tulane University of Health Sciences, New Orleans, Louisiana, USA

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      【摘要】
   We used serial analysis of gene expression to catalog the transcriptome of murine mesenchymal stem cells (MSCs) enriched from bone marrow by immunodepletion. Interrogation of this database, results of which are delineated in the appended databases, revealed that immunodepleted murine MSCs (IDmMSCs) highly express transcripts encoding connective tissue proteins and factors modulating T-cell proliferation, inflammation, and bone turnover. Categorizing the transcriptome based on gene ontologies revealed the cells also expressed mRNAs encoding proteins that regulate mesoderm development or that are characteristic of determined mesenchymal cell lineages, thereby reflecting both their stem cell nature and differentiation potential. Additionally, IDmMSCs also expressed transcripts encoding proteins regulating angiogenesis, cell motility and communication, hematopoiesis, immunity and defense as well as neural activities. Immunostaining and fluorescence-activated cell sorting analysis revealed that expression of various regulatory proteins was restricted to distinct subpopulations of IDmMSCs. Moreover, in some cases, these proteins were absent or expressed at reduced levels in other murine MSC preparations or cell lines. Lastly, by comparing their transcriptome to that of 17 other murine cell types, we also identified 43 IDmMSC-specific transcripts, the nature of which reflects their varied functions in bone and marrow. Collectively, these results demonstrate that IDmMSC express a diverse repertoire of regulatory proteins, which likely accounts for their demonstrated efficacy in treating a wide variety of diseases. The restricted expression pattern of these proteins within populations suggests that the cellular composition of marrow stroma and its associated functions are more complex than previously envisioned. # ~, t, d2 U  K
      【关键词】 Serial analysis of gene expression Mesenchymal stem cells Bone marrow stromal cells7 S- i( I" Z* G! \+ H; ^
   INTRODUCTION! q, H- w2 ~% r7 \+ l& l% c# {

Mesenchymal stem cells (MSCs) resident in adult bone marrow are best characterized by their capacity to differentiate into various connective tissue cell lineages . However, it is unclear if this heterogeneity contributes to or detracts from the potential clinical utility of MSCs.  E4 V* ~1 H$ d* W4 y- X, P% s

A large number of studies aimed at evaluating the therapeutic potential of MSCs use murine models, but MSC cultures established from mouse bone marrow are replete with hematopoietic cells, which adhere to plastic, stromal cells or the matrix molecules they secrete . Consequently, immunodepletion produces a cell population that, based on phenotype and function, more closely recapitulates properties of the bona fide MSC.# G3 a. t" l. }- P
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In this report, we used serial analysis of gene expression (SAGE) to catalog the transcriptome of IDmMSCs. Its interrogation revealed that IDmMSCs express a diverse repertoire of mRNAs, including those that reflect the developmental potential of the cells and their varied functions in bone and marrow. The latter include transcripts encoding regulatory proteins that modulate angiogenesis, bone turnover, cell motility and communication, hematopoiesis, immunity and defense as well as neural activities. Our analysis revealed that different classes of regulatory proteins are expressed within specific subpopulations of IDmMSCs. Moreover, some of these proteins are absent or expressed at reduced levels in other MSC preparations and cell lines. Collectively, these results suggest that the heterogeneity of IDmMSC populations with regard to encoded biological activities likely contributes to their demonstrated efficacy in a wide variety of disease models. Further characterization of these unique subpopulations may provide more efficacious cellular vectors tailored for treating specific diseases.: p: Q) G( M- A& z

MATERIALS AND METHODS
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Isolation of Murine MSCs8 m$ ^/ g7 _8 O! H
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Murine MSCs were isolated from bone marrow and purified by immunodepletion as previously described . Briefly, whole bone marrow purged from the long bones of FVB/N mice was cultured in alpha minimum essential medium (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal calf sera (lot no. F0091; Atlanta Biologicals, Atlanta, https:www.securewebexchange.com/atlantabio.com) and PenStrep (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) at 37¡ãC with 5% CO2 for 72 hours, and then the nonadherent cells were removed by aspiration. Cells were cultured an additional 5¨C7 days with a single media change and then harvested by gentle scraping after incubation in 0.25% trypsin and 1 mM EDTA. Cells were dispersed by gentle agitation, filtered through a 70-µm filter (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), suspended in Hanks¡¯ balanced salt solution (HBSS) at 40 x 106 cells per ml, incubated on a rotator for 1 hour at 4¡ãC, and then successively for 50-minute intervals with M-280 Dynabeads (5 beads/cell) (Dynal Biotech LLC., Brown Deer, WI, http://www.dynalbiotech.com) conjugated to anti-CD11b, anti-CD34, and anti-CD45 antibodies (10 µg/mg beads). The immunodepleted cells were diluted in 5 ml of HBSS and counted. Total RNA was prepared from approximately 1 x 106 cells using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, http://www.qiagen.com) according to the manufacturer¡¯s instructions. All RNA samples were treated with DNase I during the purification procedure to ensure removal of contaminating DNA. The cell lines D1 ORL UVA (no. CRL-12424) and M2¨C10B4 (no. CRL-1972) were purchased from the American Type Culture Collection (Manassas, VA, http://www.atcc.org) and cultured according to the manufacturer¡¯s instructions. C57BL/6 and DBA1 murine MSCs were obtained from the Center for Gene Therapy at Tulane University.1 ~4 n4 s7 B3 {3 |8 T8 V
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SAGE
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SAGE was conducted using the I-SAGE kit (Invitrogen Corporation) according to the manufacturer¡¯s instructions. Concatenated ditags were cloned into pZero and transduced into electro-competent bacteria (Invitrogen Corporation) by electorporation (BTX, Holliston, MA, http://www.btxonline.com). Bacterial colonies were screened by blue/white selection to identify those that harbored vectors containing ditags. Plasmid DNA was isolated using the Genomic DNA isolation kit (Millipore, Bed-ford, MA, http://www.millipore.com) and the Robosmart 384 automated workstation (MWG Biotech, High Point, NC, http://www.mwg-biotech.com). Plasmids were sequenced using the BigDye Terminator Cycle Sequencing Reaction kit and analyzed using a 377 ABI automated sequencer (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Sequence files were analyzed using the SAGE program group .

cDNA Library Construction

A cDNA library was constructed from the same RNA used for SAGE via the SMART® cDNA Library Construction Kit (Gibco-BRL). The resulting nonamplified library contained approximately 2.6 x 106 independent clones. After amplification, the library was aliquoted into pools of decreasing complexity to facilitate screening by polymerase chain reaction (PCR). Integrity of the cDNA library was measured using control reverse transcription (RT)¨CPCR primers for genes of varying abundance and size (Gibco-BRL). To validate SAGE tags, 50 µl of the primary pool of the amplified phage library was boiled for 5 minutes, and then aliquots (1 µl) were used as input in PCR reactions (100 µl) containing 100 pmoles of forward and reverse gene-specific primers, 1x PCR buffer, 0.2 mM dNTPs, and 0.5 U Taq polymerase (Sigma, St. Louis, http://www.sigmaaldrich.com). After an initial denaturation step at 94¡ãC for 3 minutes, reactions were amplified for 30 cycles at 94¡ãC for 30 seconds, 55¨C65¡ãC for 45 seconds, and 72¡ãC for 90 seconds, followed by a final incubation at 72¡ãC for 5 minutes. PCR product were electrophoresed through a 1% agarose gel, excised from the gel, purified using GeneElute columns (Sigma), and then cloned using the AdvanTAge PCR cloning kit (Clontech, Palo Alto, CA, http://www.clontech.com). Plasmid DNA was isolated and sequenced as described above to confirm the identity of each product.& R+ e1 @% w0 f: q$ [0 d& f

Real-Time PCR
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Total RNA (25 ng) was converted to cDNA and amplified by the PCR process using the TaqMan® EZ RT-PCR kit (Applied Biosystems) according to the manufacturer¡¯s instructions. Reactions were performed on a 7900 HT sequence detector (Applied Biosystems), and transcript levels were quantified using the relative Ct method by employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. The following Assay-On-Demand Taqman® probes (Applied Biosystems) were used for analysis: Dusp1, Mn00457274-gl; Irx3, Mm00500463-ml; Mef2d, Mm00504929-ml; Omd, Mm 00449589-ml; Spry4, Mm00442345-ml; Timp3, Mn00441826-ml; and Twist, Mm00442036-ml.
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Flow Cytometry* j; Z, R6 d. k+ b  @/ D# N

Aliquots (2.5 x 105) of IDmMSCs were suspended in 50 µl of wash buffer (0.1% sodium azide, 1.0% bovine serum albumin ) containing a rat anti-mouse CD16/CD32 antibody (Fc Block; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) at a concentration of 1 µg per 1 x 106 cells and incubated for 3¨C5 minutes at 4¡ãC in the dark. Wash buffer (50 µl) containing 5 µg of the appropriate primary antibody (BD Pharmingen) was added, and the cells were incubated for an additional 20 minutes. Cells were washed twice with 200 µl of wash buffer and, where necessary, were incubated for 20 minutes in wash buffer (100 µl) containing 5 µg of a fluorochrome-conjugated secondary antibody (BD Pharmingen). The extent of cell labeling was evaluated using a Beckman Coulter Model Epics XL flow cytometer (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). Isotype controls were run in parallel using the same concentration of each antibody tested.
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Immunostaining4 _4 [' Y7 B/ s" s* W( w& V4 d

Aliquots (2 x 104) of IDmMSCs were plated in eight-well chamber slides (BD Biosciences) and after 24¨C48 hours fixed with 2% paraformaldehyde/0.2% glutaraldehyde for 15 minutes at room temperature. Cells were then incubated for 10 minutes in blocking buffer (PBS containing 0.1% BSA, 5% Tween-20, and 20% of the appropriate animal sera), washed, and then incubated overnight at 4¡ãC with a 1:100 dilution of an anti¨CIL-15, anti-Cyr61, or anti-MIF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com). Cells were then washed, incubated with the appropriate fluorescein isothiocyanate¨Cconjugated secondary antibody for 10 minutes at room temperature, washed, and then a cover slip was applied using mounting media containing 4',6-Diamidino-2-phenylindole (DAPI). Alternatively, fixed cells were incubated in 0.3% hydrogen peroxide for 1 minute at room temperature, incubated with a 1:100 dilution of an anti-VEGFB, anti-IRX3, anti-PTN, or anti-TWIST antibody (Santa Cruz Biotechnology) followed by a biotinylated anti-goat IgG antibody, and then staining was visualized using the Tyramide Signal Amplification kit according to the manufacturer¡¯s instructions (Invitrogen Corporation). Fluorescent micrographs were obtained using a Leica RX-DMV upright fluorescent microscope (Meyer Instruments, Inc., Houston, TX, http://www.meyerinst.com) attached to a digital camera (Cooke Sensicam High Performance; Hamamatsu Corp., Bridgewater, NJ, http://www.hamamatsu.com) and rendered using Slidebook® software (Intelligent Imaging Innovations, Denver, http://www.intelligent-imaging.com).

SAGE Database Analysis

Catalogued SAGE tags were annotated in Microsoft® Access (Microsoft, Redmond, WA, http://www.microsoft.com) as described in the I-SAGE manual (Invitrogen Corporation). The resulting database was interrogated using a collection of macros we developed that run in Microsoft® Excel. Annotated SAGE databases were exported to Excel, and the hierarchical relationships embedded in the data were removed using the macro Telescope, which parcels each gene into a separate row of the spreadsheet and repeats its associated DNA tag and frequency as many times as there are associated genes. Telescope then collapses the tag and frequency information into a single bin. Descriptors for nonunique tags that match two genes are concatenated and placed in a single bin, and those matching three or more genes are abbreviated as "multiple match." Once in this format, the macro Recognize Machinery was used to interrogate the database using a list based on gene ontologies. The macros Master, Align, and Set Logic were used for comparative analysis of SAGE databases. Briefly, Master generates a master list of all unique tags contained within a specified number of databases. The macro Align places tags from each library in register with those of the master list while maintaining the register between the tag and its frequency of occurrence. Finally, Set Logic determines the union or disjunction of the libraries being analyzed and compiles the resulting information on a separate spreadsheet.

RESULTS" h& \3 K0 s0 b- h/ n( O  _5 W

Composition of the 500 Most Abundant Expressed SAGE Tags  }% |& y" j" g

Previously, we described a method based on immunodepletion to enrich MSCs from murine bone marrow (34, 35]. IDmMSCs lack expression of the hematopoietic and endothelial cell lineage markers CD11b, CD31, CD34, CD45, and CD117 but express markers typical of MSCs, including CD9, CD29, CD44, CD81, CD106, and Sca1. IDmMSCs also exhibit poor growth in vitro, and their capacity for multilineage differentiation is reversibly inhibited by FGF2. These latter characteristics distinguish the cells from other MSC cell lines and populations isolated by long-term propagation in vitro .4 y0 t9 X) A& @0 U# s- y
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To better characterize their biology, we catalogued the IDmMSC transcriptome via SAGE by sequencing 59,007 SAGE tags, 15,815 of which were unique. The most highly expressed transcript based on SAGE tag abundance was that corresponding to the ß-galactoside binding lectin galectin 1 (Lgals1), which plays a role in cell adhesion, migration, and proliferation . By screening a cDNA library generated using the same RNA as for SAGE analysis, we confirmed that catalogued SAGE tags correspond to expressed transcripts. PCR products amplified from the cDNA library were cloned and sequenced to confirm their identity (Fig. 1A).* [. }) ]+ l- M: w) }0 }

Figure 1. Validation of catalogued SAGE tags. Polymerase chain reaction products amplified using gene specific primers from an IDmMSC cDNA library were electrophoresed through a 1% agarose gel, cloned, and their corresponding sequences analyzed via BLAST to confirm their identity. (A): Expressed transcripts corresponding to the top 500 most abundant SAGE tags that map uniquely to a single mRNA. (B): Validation of a representative sampling of other expressed transcripts catalogued by SAGE. Abbreviations: Actg, actin, gamma, cytoplasmic; Agpt, angiopoietin; BLAST, Basic Local Alignment Search Tool; Ccl7, Chemokine ligand 7; Clec2d, C-type lectin domain family 2, member d; Csrp1, cysteine rich protein 1; Dsn, destrin; Dusp1, dual sensitivity phosphatase 1; Fos, FBJ osteosarcoma oncogene; Fstl, follistatin-like; Gpdh, glyceraldehyde dehydrogenase; Hdgf, hepatocyte derived growth factor; Ilk, integrin linked kinase; Il1rn, Interleukin-1 receptor antagonist; Irx3, Iroquois related homeobox 3; Jmj, Jumonji; Lgals1, galectin 1; Ly75, lymphocyte antigen 75; Mif, macrophage migration inhibitory factor; Mmp2, matrix metalloproteinase 2; Npnt, nephronectin; Nspe1, nuclease sensitive element binding protein 1; Ogn, osteoglycin; Omd, osteomodulin; Osn, osteonectin; Pfn1, profilin 1; Prdx1, peroxiredoxin 1; SAGE, serial analysis of gene expression; Six1, sine oculis-related homeobox homologue 1; Std, stanniocalcin; Tagln, transgelin; Timp3, tissue inhibitor of metalloproteinase 3; Tubb5, tubulin, beta 5; Vegfb, vascular endothelial growth factor b.
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SAGE Tags Corresponding to Mesenchymal Lineage¨CSpecific Transcripts* |' b- c% X! @+ ]+ p

Further interrogation of the IDmMSC transcriptome revealed that the cells expressed mRNAs characteristic of determined mesenchymal cell lineages. For example, 2.9% of all SAGE tags catalogued corresponded to skeletal-specific transcripts and these mapped specifically to 91 separate mRNAs (Supplemental Database 1). These included various matrix molecules such as secreted acidic cysteine rich glycoprotein (osteonectin), fibronectin, matrix Gla protein, cadherin-11, collagens type I, II, V, IX, and XI, decorin, fibromodulin, neurochondrin, and osteoglycin. Also represented were several growth factors and transcriptional regulators important for bone development and function. The former included transforming growth factor ß2 and ß3, connective tissue growth factor, insulin-like growth factor, and bone morphogenetic proteins 1, 4, and 5. The latter included the FBJ osteosarcoma gene (Fos), which is highly expressed in embryonic and adult bone tissue, as well as Twist 2, which plays a role in mesoderm specification during development .5 F' z1 b- P3 J* L+ `

In addition, approximately 1.8% of all SAGE tags corresponded to muscle-specific transcripts, and these mapped specifically to 78 mRNAs encoding contractile proteins, calcium binding proteins, calcium transporting ATPases, voltage-sensitive calcium channels, and skeletal-specific, smooth muscle¨Cspecific, and cardiac muscle¨Cspecific isoforms of myosin. Other catalogued transcripts encoded the muscle-specific proteins desmin, smoothelin, myocyte enhancer factor 2D, acidic syntrophin 1, titincap, and troponin 1, T1, and T3. Lastly, we also identified 76 transcripts mapping to unique tags encoding various cytokines, chemokines, and adhesion molecules that regulate aspects of hematopoiesis and 22 transcripts that regulate adipocyte differentiation or function. The presence of mesenchymal lineage¨Cspecific transcripts within the IDmMSC transcriptome reflects their differentiation potential as defined by functional assays. The diversity and abundance of such transcripts further suggest that mesengenesis is an active process in adherent cultures even in the absence of exogenous agents that promote cellular differentiation.
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Categorization of the IDmMSC Transcriptome Based on Gene Ontologies( ~& ^6 f7 m+ b' X# v; D: p

Using gene ontologies defined by the Celera® Discovery System Panther classifications .2 f1 \* w6 l6 k1 t% A" H

Figure 2. Categorization of the IDmMSC transcriptome via gene ontologies. Pie chart illustrating the percentage of SAGE tags that map uniquely to specific transcripts classified according to their (A) molecular function or (B) biological processes based on the Celera Panther Gene Ontology classification system. Abbreviations: IDmMSC, immunodepleted murine mesenchymal stem cell; SAGE, serial analysis of gene expression.1 _1 v' H- ~- J' z% G

Categorizing transcripts based on their biological process revealed that the majority of SAGE tags mapped to mRNAs were involved in protein modification (12.9%), followed by signal transduction (8.91%), immunity and defense (7.8%), cell adhesion (6.5%), nucleic acid metabolism (5.86%), and cell structure and motility (5.76%). The transcriptome also contained numerous transcripts encoding proteins that regulate neural activities, including various axon guidance and neural cell adhesion molecules, neurite-inducing factors, neurotransmitter receptors, neurotrophins, and a large number of proteins involved in vesicle transport, synaptogenesis, and synaptic transmission (Supplemental Database 1). Other transcripts encoding proteins common to neuronal cells or that regulate nervous system development were also identified, including the transcription factor neurogenic differentiation 1 (Neurod1), the neurofilament proteins Nef1 and Nef3, and the growth suppressor necdin, which is expressed in all postmitotic neurons in the brain . The IDmMSC transcriptome also contained mRNAs encoding proteins with proangiogenic activity, including vascular endothelial growth factor B, cysteine rich protein 61 (Cyr61), and connective tissue growth factor, as well as factors affecting endothelial cell growth and migration, such as angio-associated migratory cell protein, angiopoietin, and hepatoma-derived growth factor (data not shown). Approximately 3.24% of the SAGE tags also mapped specifically to transcripts that function in developmental processes, and 40% of these tags encoded proteins that play a role in the specification and differentiation of mesoderm (Fig. 3 and Supplemental Database 1).# C5 c* _! N# `7 C1 X
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Figure 3. Number of transcripts expressed by IDmMSCs that regulate developmental processes. The pie chart illustrates the total number of transcripts that mapped uniquely to a given SAGE tag(s) and that function in specific developmental processes. Abbreviations: IDmMSC, immunodepleted murine mesenchymal stem cell; SAGE, serial analysis of gene expression.
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Restricted Expression of Regulatory Proteins in IDmMSC Populations
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The diverse repertoire of mRNAs expressed by IDmMSCs implicates the cells in a wide array of biological activities important for the function of bone and marrow. To validate these results, we analyzed the expression profile of different classes of regulatory proteins in IDmMSC populations. Immunofluorescence staining confirmed that the cells express the cytokine interleukin-15 (IL-15), the angiogenic factors cysteine rich protein 61 [CYR61) and vascular endothelial growth factor B [VEGFB), the transcriptional regulator iroquois-related homeobox 3 (IRX3), and TWIST, as well as the multifunctional proteins pleiotrophin (PTN) and MIF. However, expression of these proteins was restricted to specific subpopulations of cells and the number of cells expressing each protein appeared highly variable. For example, whereas IL-15 and IRX3 were detected in approximately half of the population, only a small percentage of cells expressed CYR61, VEGFB, PTN, and TWIST. Furthermore, the subcellular location of IRX3 and TWIST varied between cells. IRX3 was localized exclusively in the cytoplasm of some cells (Fig. 4E) but was confined to the nucleus or evident in both compartments in other cells (Fig. 4F). TWIST showed a similar expression pattern and appeared to be highly expressed in the nucleus of a small number of cells (Fig. 4O). Fluorescence-activated cell sorting (FACS) analysis confirmed that the number of cells expressing these different regulatory proteins was highly variable within IDmMSC populations (Fig. 5).

Figure 4. Immunohistochemical localization of specific regulatory proteins to subpopulations of immunodepleted murine mesenchymal stem cells (IDmMSCs). IDmMSCs were stained with fluorescein isothiocyanate (FITC)¨Cor Cy3-conjugated antibodies specific for (A¨CC) interleukin-15 (IL15), (D¨CF) iroquois related homeobox 3 (IRX3), (G¨CI) cysteine rich protein 61 (CYR61), (J¨CL) vascular endothelial growth factor B (VEGFB), (M) pleiotrophin (PTN), (N) macrophage migration inhibitory protein (MIF), and (O) TWIST and counterstained with 4',6-Diamidino-2-phenylindole (DAPI). Arrows indicate localized expression of IRX3 (F) and TWIST (O) within the nucleus.6 d: |8 z, o# w, d8 X( D

Figure 5. Percentage of cells within different murine mesenchymal stem cell populations that express different classes of regulatory proteins. Histograms showing the percentage of immunodepleted murine mesenchymal stem cells (IDmMSCs) and C57BL/6, M210B4, and CRL-12424 cells that reacted with antibodies against interleukin-15 (IL15), cysteine rich protein 61 (CYR61), macrophage migration inhibitory protein (MIF), interleukin-1 receptor antagonist (IL1RN), and pleiotrophin (PTN).

Due to the fact that IDmMSCs undergo limited expansion in vitro prior to immunodepletion, we also anticipated that the cells may express a broader repertoire or a greater abundance of transcripts as compared with other murine MSC populations. Therefore, we compared mRNA and protein levels of different classes of regulatory proteins in IDmMSCs with those of the MSC cell lines D1 ORL UVA and M2¨C10B4, as well as MSC populations isolated from C57Bl/6 and DBA1 mice by long-term propagation in vitro .
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Figure 6. mRNA levels of different regulatory factors were higher in immunodepleted murine mesenchymal stem cells (IDmMSCs) as compared with other murine mesenchymal stem cell preparations. Total RNA prepared from IDmMSCs, C57BL/6, and DBA1 cells and the M210B4 and CRL-12424 cell lines was analyzed by real-time polymerase chain reaction to quantify expression levels of dual specificity phosphatase 1 (Dusp1), iroquois related homeobox 3 (Irx3), osteomodulin (Omd), sprouty 4 (Spry4), tissue inhibitor of metalloproteinase 3 (Timp3), and Twist 1. Transcript abundance was determined using the relative Ct method and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. All samples were run in triplicate, and the mean ¡À SD was plotted.# Q6 o+ e7 S  }7 M+ A5 k
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Molecular Fingerprint of IDmMSCs via Comparative Genomics
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To determine a molecular fingerprint for IDmMSCs, we compared their transcriptome to that of 17 different murine cell types and tissues for which SAGE databases are available in the public domain (Table 1). The 18 SAGE databases contained a total of 668,445 tags that occurred with a frequency of two or greater, and 37,479 of these were unique. A total of 575 tags were found exclusively in the IDmMSC database. Strikingly, 90 (15.6%) tags corresponded to RIKEN cDNAs, 49 (8.5%) failed to match any known gene sequences, 45 (7.8%) corresponded to uncharacterized expressed sequences, and 7 (1.2%) encoded hypothetical proteins. Therefore, approximately 33% of all SAGE tags unique to IDmMSCs as defined by the criteria outlined above encoded proteins of indeterminate function. Gene annotations corresponding to transcripts mapping uniquely to the remaining tags were filtered against all transcriptomes to remove redundancies, revealing 43 transcripts specific to IDmMSCs (Supplemental Database 1). These included the skeletal proteins osteoglycin, osteomodulin, and parathyroid hormone receptor 1, as well as chemokine ligand 7 (Ccl7) and lymphocyte antigen 75 (Ly75). Ccl7 mediates macrophage recruitment during inflammation but, after cleavage by gelatinase A, functions as a general chemokine antagonist that dampens inflammation . Consequently, the repertoire of IDmMSC-specific transcripts appears to reflect the unique functions associated with MSCs and/or their progeny, including skeletogenesis and immunity and defense.8 Q' n. _6 W! k
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Table 1. List of transcriptomes used for comparative analysis to generate a genetic footprint for immunodepleted murine mesenchymal stem cells0 S$ e8 b% E6 q7 M& J3 O

DISCUSSION! D. S) Y6 U) e; B/ t5 ?
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Our SAGE analysis is consistent with other gene profiling studies indicating that MSCs are characterized by an abundance of mRNAs encoding various connective tissue proteins . IL-1 is also a potent stimulator of bone resorption. Therefore, expression of IL-1RN together with Clec2d, a C-type lectin that inhibits multinucleate osteoclast formation, may enable IDmMSCs to regulate bone turnover, as well.
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Our SAGE analysis also provides a framework to reconcile the molecular and functional heterogeneity of MSC populations (Fig. 7). For example, previous reports have shown that MSCs enriched from marrow by plastic adherence represent a collection of cells with varying potentials that follow a deterministic differentiation program in vitro .
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Figure 7. The IDmMSC transcriptome reflects the plasticity of MSCs as well as the varied functions they and their progeny perform in bone marrow. Schematic summarizes the nature of catalogued transcripts in the IDmMSC transcriptome. These include transcripts that regulate mesoderm specification and development or that are characteristic of determined mesenchymal cell lineages, which reflect the developmental potential of MSCs. Also identified were transcripts that participate in angiogenesis, cell motility and communication, hematopoiesis, immunity and defense, as well as neural activities, which reflect the varied biological activities that MSCs and progeny participate in within bone and marrow stroma. Abbreviations: IDmMSC, immunodepleted murine mesenchymal stem cell; MSC, mesenchymal stem cell; SAGE, serial analysis of gene expression.3 S6 v& B5 h6 M/ L& J  K0 d
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Additionally, identification of transcripts regulating angiogenesis, hematopoiesis, cell motility and communication, immunity and defense, as well as neural activities reflects the characteristics and/or associated functions of stromal cell subtypes known to exist in marrow. For example, a number of specialized cell types in stroma are highly motile, including reticular cells and adventitial reticular (AR) cells. Reticular cells are so named because they extend or reticulate long, cytoplasmic processes into the hematopoietic cords, and AR cells mediate egress of hematopoietic cells into the vasculature by contracting or physically migrating to and from the sinus wall. Furthermore, motility of these cells is coordinated by communication via gap junction formation with periarterial adventitial (PAA) and hematopoietic cells .! J0 F1 w0 l, f; v* |
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Bone and marrow are also innervated by nervous tissue. Efferent nerve terminals from fibers that track into the hematopoietic cords terminate directly onto AR cells. Because the PAA and AR cells are connected by gap junctions, they are indirectly coupled to each other forming a circuit termed the "neuroreticular complex," providing a means by which nervous input can alter stromal function to regulate hematopoiesis . Co-operatively, these factors induce capillary proliferation and expansion of the sinusoidal space. Expression of these and other angiogenic factors likely modulates vessel growth and remodeling as well, processes essential for bone growth.

The large number of expressed transcripts encoding unique regulatory factors revealed by our SAGE analysis greatly expands the repertoire of biological activities that can be ascribed to stromal elements within marrow. This diversity of function is also reflected by the cohort of transcripts deemed unique to IDmMSCs, which included several small leucine-rich proteins unique to skeletal tissue and proteins that affect immune cell activity, proliferation, and chemotaxis, as well as angiogenesis. IDmMSC-specific transcripts encoding homeotic genes important in limb morphogenesis further substantiate the existence of early mesodermal progenitors or stem cells within plastic adherent populations. Comparative studies suggest that expression levels of specific regulatory proteins may be highly variable between preparations of murine MSCs. IDmMSCs express higher levels of these factors than other MSC populations, likely because long-term propagation of cells in vitro selects against specific subpopulations. Differences in the expression profile of regulatory proteins between murine MSC populations may produce disparate outcomes in both in vitro and in vivo experiments.
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SUMMARY

Our comprehensive SAGE analyses are the first to provide a molecular basis for the unique biological characteristics of murine MSC populations enriched from marrow by plastic adherence. Furthermore, the finding that IDmMSCs express a diverse array of regulatory proteins, expression of which is restricted to specific subpopulations of cells, indicates that the cellular complexity and biological functions of marrow stroma are more diverse than previously envisioned. Based on these results, we propose that the heterogeneity of IDmMSC populations likely attributes to their demonstrated therapeutic efficacy in a wide variety of disease models. Exploiting the unique characteristics of these subpopulations may provide more efficacious cellular vectors tailored for treating specific diseases.
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ACKNOWLEDGMENTS

This research was supported in part by a grant from the National Institutes of Health to D.G.P. (R01-AR44210-01A1), the Louisiana Gene Therapy Research Consortium (New Orleans), and HCA-the Health Care Company (Nashville, TN). The authors would like to thank Peggy Wolfe for providing the C57BL/6 and DBA1 murine MSCs.& \8 g+ Z0 b& o
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The authors indicate no potential conflicts of interest.
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