The Profile of Gene Expression of Human Marrow Mesenchymal Stem Cells

Authors

  • Wilson A. Silva Jr.,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Dimas T. Covas,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Rodrigo A. Panepucci,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Rodrigo Proto-Siqueira,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Jorge L.C. Siufi,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Dalila L. Zanette,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Anemari R.D. Santos,

    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
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  • Marco A. Zago M.D., Ph.D.

    Corresponding author
    1. Center for Cell Therapy and Regional Blood Center, Department of Clinical Medicine, Faculty of Medicine, Ribeirão Preto, Brazil
    • Hemocentro, R. Tenente Catao, Roxo, 2501, 14051-140 Ribeirao Preto, Brazil. Telephone: 55-16-3963-9361; Fax: 55-16-3963-9309
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Abstract

Mesenchymal stem cells (MSCs) are multipotent precursors present in adult bone marrow, that differentiate into osteoblasts, adipocytes and myoblasts, and play important roles in hematopoiesis. We examined gene expression of these cells by serial analysis of gene expression, and found that collagen I, secreted protein acidic and rich in cysteine (osteonectin), transforming growth factor beta- (TGF-β) induced, cofilin, galectin-1, laminin-receptor 1, cyclophilin A, and matrix metalloproteinase-2 are among the most abundantly expressed genes. Comparison with a library of CD34+ cells revealed that MSCs had a larger number of expressed genes in the categories of cell adhesion molecule, extracellular and development. The two types of cells share abundant transcripts of many genes, some of which are highly expressed in myeloid progenitors (thymosin-β4 and β10, fos and jun). Interleukin-11 (IL-11), IL-15, IL-27 and IL-10R, IL-13R and IL-17R were the most expressed genes among the cytokines and their receptors in MSCs, and various interactions can be predicted with the CD34+ cells. MSCs express several transcripts for various growth factors and genes suggested to be enriched in stem cells. This study reports the profile of gene expression in MSCs and identifies the important contribution of extracellular protein products, adhesion molecules, cell motility, TGF-β signaling, growth factor receptors, DNA repair, protein folding, and ubiquination as part of their transcriptome.

Introduction

In addition to hematopoietic cells, bone marrow (BM) comprises a heterogeneous population of cells that plays a key role in hematopoiesis, referred to as marrow stromal cells, including endothelial cells, adipocytes, osteoblasts and fibroblasts. Mesenchymal stem cells (MSCs) are multipotent precursors present in adult BM, capable of differentiating into osteoblasts, adipocytes, and myoblasts [13]. Although they represent only about 0.01%-0.001% of the marrow cells, they can be separated from the hematopoietic stem cells (HSC) because they adhere to glass and plastic [4]. Once in culture they proliferate to originate spindle-shaped cells in confluent cultures, but exhibit a variable morphology and differentiation potential under appropriate conditions. These cells are present in adult BM and peripheral blood, and in the fetal BM and liver [5]. Besides their ability to give rise to cells that constitute the BM stroma, they have been reported to originate glial and neuronal cells, whereas protein and mRNA expression have demonstrated epithelial, endothelial, and neuronal markers. A mesoderm progenitor that gives rise to mesenchymal and to endothelial cells has been identified [6]. Although part of these diverse results is probably caused by the fact that the majority of studies have dealt with heterogeneous cell populations that differ because of the preparation protocols or the time in culture, Tremain et al. have demonstrated that markers of various differentiation lineages are concomitantly present in a colony derived from a single MSC [7] providing evidence for their stem-cell (SC) nature.

The wide therapeutic potential of these cells has attracted much attention to them [2, 8], and in vitro and in vivo functional studies and therapeutic trials have been started [9]. However, the transcriptome and broad gene expression profile of a well-defined MSC population has not been described in detail. In addition to the study of Tremain et al. [7], the reports have been limited to analyzing the expression of gene families under particular experimental conditions. We have employed serial analysis of gene expression (SAGE) to examine the gene expression of MSC obtained from normal human BM and compared it with the expression profile of CD34+ hematopoietic precursors.

Materials and Methods

Isolation and Culture of Human MSCs

Human MSCs were obtained by aspiration from the iliac crest of a BM donor who gave consent after full information. The mononuclear cells were separated by ficol gradient (Ficoll-Paque; Amersham Biosciences; Peapack, NJ; http://www.bioprocess.amershambiosciences.com), washed in Hank's balanced salt solution, and then cultured in a 25 cm2 tissue culture flask (Falcon; BD Biosciences; Franklin Lakes, NJ; http://www.bdbiosciences.com) in alpha-minimal essential medium supplemented with 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 20% fetal calf serum (Atlanta Biological; Norcross, GA; http://www.atlantabio.com) [10, 11]. After 24 hours the nonadherent cells were removed and the adherent layer cultured until it reached 50%-70% confluence. Cells were then harvested by incubation with 0.2% trypsin-EDTA. Cells collected after the fourth culture passage were stimulated to osteoblast and to adipocyte differentiation as described [2, 10] and used for RNA extraction for SAGE analysis.

Flow Cytometry Analysis

The cells harvested were washed in phosphate-buffered saline, counted pelleted by centrifugation, and resuspended in 100 μl of the appropriate monoclonal antibody and corresponding isotype controls (Pharmingen; San Diego, CA; http://www.bdbiosciences.com/pharmingen). The labeled cells were analyzed on a FACSort by collecting 10,000 events with the Cell Quest software program (Becton Dickinson; San Jose, CA; http://www.bd.com). The antibodies used were CD90-PE, CD51/61-PE, CD29-PE, CD49e-PE, CD49d-PE, CD44-FITC, CD45-FITC, CD13-FITC, HLADR-FITC, HLAclassI-FITC.

SAGE Procedure

Total RNA was prepared from 4 × 107 cells obtained from a fresh cell culture using TRIzol®LS Reagent (Cat No. 10296010; Invitrogen Corporation; Carlsbad, CA; http://www.invitrogen.com) and treated with RQ1 RNase-Free Dnase (Cat. No. M6101; Promega Corporation; Madison, WI; http://www.promega.com) according to manufacturer's instructions. Absence of DNA contamination was ascertained by Southern blot analysis with a mitochondrial DNA marker (D-loop) as a probe, using the treated RNA as template in a polymerase chain reaction (PCR). Thirty μg of total RNA were then used for the SAGE procedure. SAGE was carried out using the I-SAGE Kit (Cat. No. T5001-01; Invitrogen) based on the original SAGE [12]. Amplified inserts were sequenced with forward M13 primer in a MegaBACE1000 sequencer and the DYEnamic ET Dye Terminator Sequencing Kit (Cat. No. US81090; Amersham Biosciences; Piscataway, NJ; http://www.amershambiosciences.com).

SAGE Analysis

Tag frequency tables were obtained from sequences by the SAGE analysis software, with minimum tag count set to one, maximum ditag length set to 28 bp, and other parameters set as default. The annotation was based on two specific tools, SAGEmap (http://www.ncbi.nlm.nih.gov/SAGE/) and CGAP SAGE Genie (http://cgap.nci.nih.gov/SAGE). We downloaded a SAGE library of CD34+ HSCs purified from BM [13] available as supplemental material in the Proceedings of the National Academy of Science (PNAS) website http://www.pnas.org. When the two libraries were compared, the number of tags was normalized to a total count of 200,000 tags.

Semiquantitative Evalution by Real Time-PCR (RT-PCR)

Total RNA was obtained from seven human tissues. The transcription reaction was performed with 2 μg of total RNA, 0.5 μg of Oligo (dT) primer and 200 U of Superscript II Rnase H Reverse Transcriptase (Invitrogen) in a total volume of 20 μl, and one-tenth of the volume of the cDNA was used in the semiquantitative PCR. The specific primers used are listed in Table 1. When the reaction was positive in the undiluted samples, the cDNA was serially diluted (1:2 to 1:128) before performing the PCR. Secreted protein acidic and rich in cysteine (SPARC) expression was measured by RT-PCR with the Taqman approach (Applied Biosystems; Foster City, CA; http://www.appliedbiosystems.com).

Table Table 1.. Sequences of the primer used for RT-PCR amplification of selected genes to corroborate the results obtained by SAGE
UniGenePrimerSequence
172928Col1A1-F5′-CGCTACTACCGGGCTGATGAT
 Col1A1-R5′-GTCCTTGGGGTTCTTGCTGATGTA
179573Col1A2-F5′-AGGGCAACAGCAGGTTCACTTACA
 Col1A2-R5′-AGCGGGGGAAGGAGTTAATGAAAC
382367Lgal1S-F5′-CCACGGCGACGCCAACACCAT
 Lgal1S-R5′-TGGGCTGGCTGATTTCAGTCAAAG
297753VIM-F5′-TCTATCTTGCGCTCCTGAAAAACT
 VIM-R5′-AAACTTTCCCTCCCTGAACCTGAG
401448TPT1-F5′-ATCCAGATGGCATGGTTGCTCTAT
 TPT1-R5′-TGCCTCCACTCCAAATAAATCACA
433399TAGLN-F5′-CTTTGGGCAGCTTGGCAGTGACCA
 TAGLN-R5′-CCAGCCCGCTTCTCCCTGCTTAG
406504TAGLN2-F5′-AGCGGACGCTGATGAATCTGG
 TAGLN2-R5′-TGGCTATGGGGAAGGGAATGTATT
111301MMP2-F5′-CAGGCACTGGTGTTGGGGGAGAC
 MMP2-R5′-CCATCGCTGCGGCCAGTATCAGTG
380674ANXA2-F5′-GGTCTCCCGCAGTGAAGTGGACAT
 ANXA2-R5′-GGCCAGGCAATGCTTAGGCAACTA
416073S100A8-F5′-GAATTTCCATGCCGTCTACAGG
 S100A8-R5′-GCCACGCCCATCTTTATCACCAG
169476GAPDH-F5′-TTAGCACCCCTGGCCAAGG
 GAPDH-R5′-CTTACTCCTTGGAGGCCATG
444446ZNF287-F5′-CATGGATGGTGATAAAAGAAA
 ZNF287-R5′-TGAGACATGATTGAAGGTTGC
406409MYO1F-F5′-CCAAGCCCCAGCCTCGGACACAT
 MYO1F-R5′-GGCGGGCGAAAGAGAAGGCAGTAT
79877MTMR6-F5′-AAGACTTATTCCCTGTGG
 MTMR6-R5′-ATTGTTTATTTTGCTCATTC
108885COL6A1-F5′-CAAGAGCCTGCAGTGGATGG
 COL6A1-R5′-CTGGTCTGAGCCTGGGATGAA
238928HT002-F5′-GGCCACCTCTAAGACCACACTGAC
 HT002-R5′-GCCGGCCATTCTAGCGATTTG
75511CTGF-F5′-GCCCGGGAAATGCTGCGAGGAGT
 CTGF-R5′-TGCAGGAGGCGTTGTCATTGGTAA
181163HMGN2-F5′-CTGGCAAGGAGGGGAATAACC
 HMGN2-R5′-GGGAGCCAAGAGGAAGTC
111779SPARC-F5′-ACAAGCTCCACCTGGACTACATC
 SPARC-R5′-GGGAATTCGGTCAGCTCAGA
 SPARC-probe5′-TTGCAAATACATCCCC

Results

Characteristics of the MSC Population

The cells that assumed a spindle-shaped morphology in confluent wave-like layers at 7 to 14 days of culture were CD13+, CD29+, CD44+, CD45, CD49d, CD49e+, CD90+, CD51/61, HLAClassI+, and HLADR. When cultured with dexamethasone and ascorbic acid, they underwent osteogenic differentiation, as demonstrated by positive calcium staining by the von Kossa reaction, whereas in culture with insulin, dexamethasone and indomethacin, they originated adipocytes, identified by vacuoles that stained positively with Sudan III. They thus have the distinguishing characteristics of the MSC [2].

Gene Expression of MSC

A total of 102,796 tags were obtained by sequencing. Excluding redundancy, these results correspond to 34,649 unique tags, 22,343 of which matched known genes or expressed sequence tags (ESTs) in the CGAP SAGE Genie mapping (84,364 total tags corresponding to 15,167 UniGene clusters), whereas 12,306 unique tags were no matches (18,432 total tags). The 50 most abundant transcripts are listed in Table 2. Some are known to be highly expressed genes in this type of cell, whereas others are recognized here for the first time.

Table Table 2.. List of the 50 most abundant tags in mesenchymal cell SAGE library
  1. a

    *Non-normalized counts (actual numbers of reads)

  2. b

    **Bold: UniGene cluster annotated by CGAP that coincides with the cluster with the higher score or the single cluster in the SAGEmap annotation. Underlined: gene expression validated by RT-PCR or real time PCR. ?: the cluster indicated was not found.

TagCount*UniGene Cluster Hs. Descriptions**
  CGAPSAGEmap 
TGGAAATGAC1319172928172928, 444446Collagen type I alpha 1, ESTs weakly similar to zinc finger protein ZNF287
ATGTGAAGAG770111779111779, 406409Secreted protein acidic cysteine-rich (osteonectin), Myosin IF
GTGTGTTTGT737118787118787Transforming growth factorβ-induced
CCCATCGTCC708294286EST, No match
GAAGCAGGAC628180370180370Cofilin 1 (non-muscle)
TACCATCAAT620169476169476, 79877Glyceraldehyde-3-phosphate dehydrogenase, Myotubularin related protein 6
GAGGGAGTTT5837606476064, 356342Ribosomal protein L27a, ESTs highly similar to S55914 ribosomal protein L27a
GCCCCCAATA576382367382367Galectin 1
CTGGGTTAAT569298262298262, 97457Ribosomal protein S19, ESTs
GGGCTGGGGT56043020790436, 430207Ribosomal protein L29, Sperm associated antigen 7
GCATAATAGG489431927431927, 356482Ribosomal protein L21, ESTs highly similar to 2113200B ribosomal protein L21
TTGGTCCTCT419356795356795, 427046Ribosomal protein L41, E1BP1 pseudogene mRNA sequence
GAAAAATGGT411181357181357, 356267Laminin receptor 1, ESTs moderately similar to RSP4_HUMAN 40S ribosomal protein SA (P40)
TAGGTTGTCT376401448401448Tumor protein translationally controlled 1
CCTAGCTGGA374401787401787Peptidylprolyl isomerase A (cyclophilin A)
TTTGGTTTTC372179573179573, 21431Collagen type I alpha 2, Suppressor of fused homolog (Drosophila)
TGTGTTGAGA369422118422118, 356428Eukaryotic translation elongation factor 1 alpha 1, mRNA expressed only in placental villi, clone SMAP83.
GGAAATGTCA359111301111301Matrix metalloproteinase 2 (gelatinase A)
TTGCTGACTT349108885108885, 238928Collagen type VI alpha 1, HT002 protein hypertension-related calcium-regulated gene
AGCACCTCCA3387530975309Eukaryotic translation elongation factor 2
TGTACCTGTA337334842334842, 249922Tubulin alpha ubiquitous, EST
TTGGGGTTTC334418650418650, 356581Ferritin heavy polypeptide 1, ESTs highly similar to ferritin heavy chain
TTCATACACC327330871?, No match
GGCTGGGGGC321408943408943, 352407, 265848profilin 1, Chromosome 1 amplified sequence 3, Phosphodiesterase 4D interacting protein (myomegalin)
TGCACGTTTT320169793169793ribosomal protein L32
ACTTTTTCAA307328511133430, 156814?, ESTs, KIAA0377 gene product
TCCAAATCGA290297753297753Vimentin, Ribosomal protein large P2
GTGTGTTGTA285422118Eukaryotic translation elongation factor 1 alpha, No match
ATCTTGTTAC280287820287820Fibronectin 1
TAATAAAGGT278399720399720, 355986Ribosomal protein S8, ESTs highly similar to S25022 ribosomal protein S8
TAAGGAGCTG277299465299465, 50651Ribosomal protein S26, Janus kinase 1 (a protein tyrosine kinase)
TTTGCACCTT2697551175511, 181163Connective tissue growth factor, High-mobility group nucleosomal binding domain 2
AGGGCTTCCA268412900412900, 356769Ribosomal protein L10, ESTs moderately similar to RL10_HUMAN 60S ribosomal protein L10
GGCAAGCCCC262425293425293, 187577Ribosomal protein L10a, SRY (sex determining region Y)-box 21
GGATTTGGCC258297753297753Ribosomal protein large P2
GATGAGGAGA247179573179573, 426977Collagen type I alpha 2, Ribosomal protein L19
CTAGCCTCAC2441437614376, 356575Actin gamma 1, ESTs
GGGAAGCAGA24112284F11 receptor, No match
ATAATTCTTT238539539, 356463Ribosomal protein S29, Farnesyltransferase CAAX box alpha
GGAGTGTGCT23196159615Myosin light polypeptide 9 regulatory
CACCTAATTG228301104?, No match
TTGGTGAAGG2287596875968, 426138Thymosin beta 4 X chromosome, promyelocytic leukemia cell mRNA (clones pHH58 and pHH81)
GTTGTGGTTA2194851648516, 99785Beta-2-microglobulin, cDNA: FLJ21245 fis clone COL01184
TCAGATCTTT212389933389933, 85112Ribosomal protein S4 X-linked, Insulin-like growth factor 1 (somatomedin C)
ACAGGCTACG209433399433399Transgelin
GGACCACTGA209119598119598, 300622Ribosomal protein L3, Plexin A2
TTCAATAAAA207424299424299, 2012Ribosomal protein large P1, Transcobalamin I (vitamin B12 binding protein, R binder family)
CCAGAACAGA205334807334807Ribosomal protein L30
CAATAAATGT202337445337445, 446130Ribosomal protein L37, ESTs
TACCTGCAGA199416073416073S100 calcium binding protein A8

A list of all the tags found in the MSC can be found at our website http://bit.fmrp.usp.br/msc_tags/.

Comparison of MSC with CD34+ Cells

The 1,000 most abundant tags of each of the two types of progenitor cells (our MSC library and the downloaded library obtained from CD34+ cells) were compared directly with the complete list of tags of the other cell type. This comparison revealed 607 tags exclusively expressed in CD34+ hematopoietic precursors, 602 exclusively expressed in MSCs and 791 tags common to both, 393 of which were more expressed in CD34+ cells and 398 more expressed in MSCs (Table 3). A search of gene ontology (GO) terms was performed for 549 and 489 unique tags among the 1,000 more expressed respectively in MSCs and CD34 cells. The search revealed that MSCs, as compared with CD34+ cells, had a higher percentage of genes in the categories of “cell adhesion” (6.1% × 1.6%), “extracellular” (11.1% × 2.9%) and “development” (11.4 × 7.3%) (p < 0.05). When compared with the number of the gene products annotated under a specific term for the whole GO, MSCs had a higher percentage of genes in “cell adhesion” (0.4% × 6.1%), “extracellular” (6.5% × 11.1%), “cell motility” (1.97% × 4.0%), and “metabolism” (48.7% × 65.0%). A comparison of the two types of cells concerning genes expressed for cell adhesion, extracellular, and motility is shown in Table 4.

Table Table 3.. Comparison of MSC and CD34+ marrow cell gene expression, as measured by the number of tags of the corresponding genes (ribosomal proteins excluded)
  1. a

    1Tags for these transcripts were at least 50 times more abundant in the MSC as compared with CD34.

  2. b

    2Tags for these transcripts were at least 50 times more abundant in the CD34 as compared with MSC.

  3. c

    3Tags correspond to at least 0.05% of total tags from one of the cells, and the relative abundance in one type of cell does not exceed 50 times the other.

Exclusively or highly represented in MSC1
  • collagens type I and type VI, SPARC, matrix metalloproteinase 2 (gelatinase), transforming growth factor β1 induced, eukaryotic translation elongation factor 1 alpha 1, fibronectin 1, light peptide 9 of myosin, transgelin, calgranulin A, heat shock protein 47, latent transforming growth factor β binding protein 2, gap junction protein alpha 1, biglycan, annexin A2, IGF binding proteins 4 and 6, hemeoxygenase 1, tropomyosin 2, connective growth factor, brain abundant membrane attached signal protein 1, galectin 1, nexin, integrins alpha 2 and alpha V, endocytic receptor, CD151 antigen

Exclusively or highly represented in CD34+stem cells2
  • CD2 antigen, kinesin family member 5, MAP2K3, MHC class II DR alpha, eukaryotic translation elongation factor 3, aldolase C fructose-biphosphate, pulmonary surfactant associated protein C, glucose phosphate isomerase, myeloperoxydase, phosphatic acid phosphatase type 2A

Highly represented in both types of cells3
  • filamin alpha, early growth response, annexin 2 ligand (calpactin I), serotonin receptor 1 D, calcyclin, calgizzarin, cofilin 1, COX8, tropomyosin 1, 5′3′ nucleotidase, benzodiazepine receptor, IGF binding protein 7, cysteine and glycine rich protein 1, vigilin, MAP2K2, pyruvate kinase, phosphoglycerate mutase 1, integrin-linked kinase, cyclophilin A and B, vimentin, thymosin β10, milk fat globule EGF 8 protein, zyxin, heavy polypeptide 1 of ferritin, glyceraldehyde-3-phospate dehydrogenase, heat shock 70 kD, light peptide 6 of myosin, high motility group protein 1, Erb-b3, CD74 antigen, cell division cycle-2 like 5, 5′-nucleotidase, transmembrane gamma-carboxyglutamic acid protein 4, B-cell CLL/lymphoma 7A

Table Table 4.. Comparison of genes expressed in MSC and in CD34+ cells for selected terms of gene ontology. Derived from the 1,000 most abundant tags in each type of cell.
    MSC:Laminin receptor 1, Integrin β1 (fibronectin receptor), Integrin alpha V (vitronectin receptor), Collagens (type I alpha 1, type III alpha 1, type IV alpha 1, type V alpha 1, type VI alpha 1, type VI alpha 2, type VI alpha 3, type VII alpha 1, type XVI alpha 1), Transforming growth factor β-induced, Connective tissue growth factor, Chondroitin sulfate proteoglycan 2 (versican), Fibronectin 1, Activated leukocyte cell adhesion molecule (ALCAM), Milk fat globule-EGF factor 8 protein, Lysyl oxidase-like 2, MIC2, CD151 antigen, RAC1, RAB13, Protein tyrosine kinase 7, Ninjurin 1, Vinculin, Osteoblast specific factor 2, Syndecan 2, Zyxin, Cadherin 11
    CD34:Laminin receptor 1, Collagen type I alpha 1, CD164 antigen (sialomucin), Selectin L, Ninjurin 2, Selectin L, Macrophage erythroblast attacher, Integrin cytoplasmic domain-associated protein 1, Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4
    MSC:Collagens (type I alpha 1, type III alpha 1, type IV alpha 1, type V alpha 1, type VI alpha 1, type VI alpha 2, type VI alpha 3, type VII alpha 1, type XVI alpha 1), SPARC (osteonectin), Insulin-like growth factor binding proteins 3, 4 and 6, Biglycan, Fibrillin 1, Fibronectin 1, Lysozyme, Macrophage migration inhibitory factor, Calgranulins A and B, Stanniocalcin 2, Lumican, Chondroitin sulfate proteoglycan 2 (versican), Granulin, Prosaposin, Connective tissue growth factor, 5-hydroxytryptamine receptor 1D, Tissue factor pathway inhibitor 2, Transforming growth factor β1, Lysyl oxidase-like 1 and 2, Follistatin-like 1, Nucleobindin 1, Matrix metalloproteinases 2 and 19, Galectin 3, Transforming growth factor β-induced, Dickkopf homolog 3, Amyloid β (A4) precursor protein, Tissue inhibitor of metalloproteinase 1 and 3, Microfibrillar-associated protein 2, Tumor protein translationally controlled 1, Cysteine-rich angiogenic inducer 61, PRSS11 (IGF binding), CRI1, ACPP, TM4SF7
    CD34:Collagen type I alpha 1, Tumor protein, translationally controlled 1, Prosaposin, Calgranulin B, Nucleobindin 2, Lysozyme, Macrophage migration inhibitory factor, Tissue factor pathway inhibitor 2, Chorionic somatomammotropin hormone 1, Surfactant, pulmonary-associated protein C, DEF6, Ribonuclease RNase A family 2, Chondroitin sulfate proteoglycan 6 (bamacan)
    MSC:Defensin alpha 1, Actins alpha 1, alpha 4 and β, Actin related protein 2/3 complex subunit 1B and subunit 2, Tumor necrosis factor receptor superfamily member 12 A, Annexin A1, Crystallin alpha B, Connective tissue growth factor, Moesin, Gap junction protein alpha 1, RAC1, Tropomyosin 2, Tropomyosin 1, Calgranulin A, Filamin A alpha, Fibronectin 1, Aldolase A
    CD34:Selectin L, Poly(A) binding protein nuclear 1, RalA binding protein 1, Crystallin alpha B, Aldolase A, Actin β, Annexin A1, Actin related protein 2/3 complex subunits 1B and 2, Nebulin-related anchoring protein, Carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4

Corroboration of SAGE Results

Gene expression was measured semiquantitatively by RT-PCR in seven different tissues (MSCs, a sample of partly purified CD34+ cells [66% purity], bulk normal BM, brain, skeletal muscle, leukocytes and liver) for the following 11 genes: COL1A1, COL1A2, matrix metalloproteinase 2 (MMP2), TPT1, LGAL1S, TGLN, TGLN2, SPARC, vimentin (VIM), ANXA2, and S100A8. The results correlated with gene expression profiles derived from SAGE data obtained from the SAGEmap site for 10 of the 11 genes tested (the only exception was S100A8). Expression of collagen type 1, alpha 1, and alpha 2, transgelin and MMP2 was detected only in MSCs, even when MSC RNA was diluted to 1:128. Except for tumor protein translationally controlled 1 (TCTP), the other genes tested were more markedly expressed in MSC. For instance, galectin-1 was at least 10 times more expressed in MSCs than in the other tissues evaluated. RT-PCR for galectin-1 was positive in MSC RNA diluted 1:128 times (1,125 tags); it was positive up to the 1:8 dilution in CD34 cells (16 tags) and liver (27 tags), up to the 1:16 dilution for bulk BM (36 tags) and brain (61 tags), and up to 1:64 dilution for peripheral blood leukocyte (92 tags) and skeletal muscle (115 tags). Similar results were obtained for VIM, annexin 2, transgelin 2, and TCTP 1. Figure 1 exemplifies these results. Additionally, for five unique tags for which there were two possible SAGEmap annotations, RT-PCR with specific primers confirmed the CGAP annotation: COL1A1 × ZNF287, SPARC × MYO1F, GAPD × MTMR6, COL6A1 × HT002, and connective tissue growth factor (CTGF) × high mobility group protein-N2 (HMGN2).

Figure Figure 1..

Semiquantitative evaluation of mRNA abundance by RT-PCR.Total RNA was diluted 1/2 to 1/128 (only 1/8 to 1/64 dilutions are shown), reverse transcribed into cDNA and then a 30-cycle PCR with specific primers located in different exons was performed. At the left is shown the reaction for vimentin (VIM) and at the right the reaction for galectin 1 (LGAL1S). MSC: mesenchymal stem cells; CD34: CD34+hematopoietic progenitor cells; Liver: adult human liver; PBL: peripheral blood leukocytes; Brain: control human normal brain (temporal); Muscle: skeletal muscle; BM: normal human unfractioned bone marrow. A control with GAPDH primers was carried out and gave positive results up to 1/128 dilution for all samples (last two columns).

Discussion

The MSCs and the HSCs are mesoderm-derived and considered to belong to two independent differentiation pathways, each with its own precursor, although there is evidence for a common precursor or for trilineage hematopoietic recovery of totally irradiated dog transplanted with CD34 fibroblast-like SCs [1416]. MSCs have been demonstrated to engraft after transplantation, with partial correction of osteogenesis imperfecta [17, 18]. Preliminary results of co-transplantation with HSCs suggest a faster engraftment and a lower incidence of graft-versus-host disease [19, 20].

The transcriptome of MSC reveals both significant differences and similarities with the CD34+ hematopoietic precursor. One-third of the most abundant gene products of one cell type is also detected in the other, while about two-thirds are exclusively or significantly overexpressed in one type of cell. Most of the highly expressed genes in MSC are related to extracellular components, receptors to matrix components, and cell adhesion molecules (CAMs), such as collagens, SPARC, galectin-1, laminin receptor, fibronectin and MMP2. SPARC is also found in fibroblasts, in cells derived from the MSC (osteoblasts and condrocytes) [21], and in cells derived from hematopoietic precursor (megakaryoblast and platelet). Galectin-1 (β-galactoside binding protein) is involved in regulation of cell adhesion, cell proliferation, and cell death of T-cells [22], B-cells [23], and the muscular differentiation of dermal fibroblasts [24]. Transforming growth factor beta (TGF-β)-induced is the third most abundant transcript, thus confirming the important role of the TGF-β signaling pathway in this cell population [25, 26], although only two tags specific for endoglin have been detected. The finding that activin A (a cytokine of the TGF-β superfamily) and its receptors are expressed moderately in the MSC agrees with the suggestion that it may influence the growth of stromal cells in an autocrine fashion, whereas only activin receptors were found in CD34 cells [27].

A comparison of Gene Ontology (GO) [28] terms between the two libraries and with all gene products in GO revealed that the number of genes expressed in the categories of CAMs and metabolism are over-represented both in MSCs and CD34. Those in the categories of extracellular, cell motility, and cell proliferation are over-represented in MSCs, and genes in the categories of extracellular and development are under-represented in CD34 cells. The most expressed adhesion molecule in the two types of cells is laminin-1 receptor, suggesting that it may contribute to colocalization of the cells in postnatal BM, in addition to other adhesion molecules that may have a homing function, such as CD44 (H-CAM), CD47, and integrins alpha 4 and alpha E. Also highly expressed in MSCs are the genes for integrin (alpha V component of vitronectin receptor and alpha 2 component of VLA or glycoprotein I/II), CD151 antigen TGF-β-induced, osteoblast specific factor 2, milk fat globule-epidermal growth factor (EGF) 8 protein (also known as medin and lactadherin), and activated leukocyte cell adhesion molecule (ALCAM). Some of these molecules, such as laminin receptor and integrins, participate also in cell surface signaling. The most striking difference between the top expressed genes of the two types of SCs is the number of genes related to cell adhesion and extracellular component.

There are also various similarities between MSCs and CD34+ cells, which include specialized genes such as filamin, calpactin, calcyclin, cofilin, insulin-like growth factor-binding protein 7, VIM, prosaposin, lysozyme and macrophage migration inhibitory factor. Abundant transcripts were found in the two cell types for genes that are highly expressed in myeloid progenitors (CD15+) [29], such as thymosin-β4 and thymosin-β10, which are involved in the differentiation of granulocytes, monocytes and lymphocytes. Recently Tsai and McKay associated nucleostemin with cell-cycle progression in stem and cancer cells [30]. This protein is present in nucleoli of nervous and embryonic stem cells, in several cancer cell lines, and is preferentially expressed in other SC-enriched populations We found transcripts for its gene in the two cell types (18 tags in MSC and 10 tags in CD34+ cells).

Cytokine and growth factor signaling is an important determinant of the functional state of these cells and of the relationship between MSC and CD34+ progenitors. We found 30 unique tags for ILs, their receptors, and related proteins that were enriched in the two progenitors cells: 10 were shared by the two types of cells, whereas 10 were exclusive of MSC and 10 were exclusive of CD34. The most abundant transcript in this category was that for the IL-10 receptor both in CD34+ and MSCs. IL-1 is produced by the CD34+ cells, whereas MSCs have moderate expression of genes for IL-1 receptor and IL-1 receptor-associated kinase 1. Three IL genes were actively expressed in MSC—IL-11, IL-15 and IL-27—whereas CD34 cells have receptors for IL-11. Other IL receptors detected in MSCs are those for IL-9, IL-13 and for IL-17, which was found also in CD34 and plays a role in hematopoietic regulation. There were also 669 tags for various growth factors, such as stem cell growth factor, TGF-β1, CTGF, hepatoma-derived growth factor, midkine (neurite growth-promoting factor 2), fibroblast growth factor 2, platelet derived growth factor C, and endothelial cell growth factor 1.

Finally, we found at least 6,300 tags related to genes from six of the seven categories indicated by Ramalho-Santos et al. [26] as basic characteristics of “stemness”: A) Notch, Yes, JAK/STAT and TGF-β pathways; B) seven genes related to interaction with the extracellular matrix; C) ubiquination pathways, protein folding, and DNA repair; D) cell cycle and cell cycle control; E) DNA helicases and histone deacetylases, and F) RNA helicases. The strategy of comparing unfractionated BM cells with the mesenchymal and hematopoietic progenitor cells (results not shown) did not reveal a common set of transcripts enriched in the more primitive cells. These findings seem to strengthen the suggestion that although some similar genes may be active in more than one SC type, there is not a rigid pattern that can be associated with the signature of “stemness” for all the SCs, since related but not identical genes may perform the same function in different SCs, and “stem” or progenitor cells of different tissues probably do not have an equivalent collection of expressed genes.

Thus we report the profile of gene expression in MSC from adult BM in culture and find both similarities and differences with CD34+ progenitors. Although the majority of the results probably reflect the gene expression inherent to this particular cell type, we cannot rule out the possible effect of culture-induced changes on gene expression. The study identifies the important contribution of extracellular protein products, adhesion molecules, cell motility, growth factor receptors, DNA repair, protein folding, and ubiquination as part of the transcriptome of these cells. However, when extrapolating these results to MSCs of other origins, it is necessary to take into consideration possible differences that depend on the anatomical site of the cell [31]. Our results must be viewed from the perspective that large-scale gene expression profiles are more adequate to propose the rationale for future hypothesis-driven studies than to provide a direct explanation for the cell functioning and behavior [32].

Acknowledgements

The authors would like to thank Maristela Orellana, Amelia G. Araujo, Marli H. Tavela, Cristiane A. Ferreira, Fernanda G. Barbuzano, and Adriana A. Marques for their assistance with the laboratory techniques, and Israel T. Silva, Marco V. Cunha, and Daniel G. Pinheiro for their help with the bioinformatic analysis.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Financiadora de Estudos e Projetos (FINEP), Brazil.

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