First published online October 4, 2007
Stem Cells
Vol. 26 No.
1
January 2008, pp.
193
-201
doi:10.1634/stemcells.2007-0524; www.StemCells.com
© 2008 AlphaMed Press
OPEN ACCESS ARTICLE
TISSUE-SPECIFIC STEM CELLS |
Human Multipotent Stromal Cells Undergo Sharp Transition from Division to Development in Culture
Benjamin L. Larson,
Joni Ylöstalo,
Darwin J. Prockop
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana, USA
Key Words. Multipotent stromal cells • Microarray • Mesenchymal stem cells • Transcriptome • Transplantation
Correspondence: Darwin J. Prockop, M.D., Ph.D., Center for Gene Therapy, Tulane University Health Sciences Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA. Telephone: 504-988-7711; Fax: 504-988-7710; e-mail: dprocko{at}tulane.edu
Received July 3, 2007;
accepted for publication September 21, 2007.
First published online in STEM CELLS EXPRESS October 4, 2007.
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ABSTRACT
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Human mesenchymal stem cells, or multipotent stromal cells (MSCs), are of interest for clinical therapy, in part because of their capacity for proliferation and differentiation. However, results from clinical trials and in vitro models have been variable, possibly because of MSC heterogeneity and a lack of standardization between MSC in vitro expansion protocols. Here we defined changes in MSCs during expansion in vitro. In low-density cultures, MSCs expand through distinct lag, exponential growth, and stationary phases. We assayed cultures of passage 2 human MSCs from three donors at low density (50 cells per cm2) at approximately 5% confluence on day 2 and after the cultures had expanded to approximately 70% confluence on day 7. On day 2, genes involved in cell division were upregulated. On day 7, genes for cell development were upregulated. The variations among three donors were less than the variation within the expansion of MSCs from a single donor. The microarray data for selected genes were confirmed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and FACScan analysis. Approximately 50% of cells at day 2 were in S-phase compared with 10% at day 7. The results demonstrated major differences in early and late stage cultures of MSCs that should be considered in using the cells in experiments and clinical applications.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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There has been considerable interest in both the biology and therapeutic potentials for the stem/progenitor cells from bone marrow referred to initially as fibroblastoid colony-forming units [1]. The cells were subsequently used as feeder layers for hematopoietic stem cells and referred to as marrow stromal cells [2], then as mesenchymal stem cells [3]. Most recently, the names multipotent mesenchymal stromal cells and multipotent stromal cells (MSCs) have been suggested [4]. Owen and Friedenstein [1, 5] isolated the cells by their ready adherence to tissue culture surfaces and expanded them as confluent cultures. Similar protocols were used by most subsequent investigators.
MSCs for clinical trials were first used in treating osteogenesis imperfecta and subsequently in mucopolysaccharidoses, graft-versus-host disease, myocardial infarction, and other diseases [6–9]. Also, the cells were tested in a large number of animal models for diseases. However, the lack of molecular data that characterize MSCs has made it difficult to evaluate results obtain with different preparations of the cells. MSC cultures are heterogeneous populations even when generated as single-cell-derived colonies [10, 11]. The rate at which MSCs expand in culture is dependent on seeding density, but distinct lag, log, and stationary phases are observed at a broad range of seeding densities [12].
To further define the molecular characteristics of the cells, we plated human MSCs from bone marrow aspirates from three different donors at low density and examined the changes in the transcriptome as the cultures expanded. As the cells expanded, the focus of the transcriptome changed from division to development.
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MATERIALS AND METHODS
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Isolation and Cultures of Human MSCs
Human MSCs were obtained from the Tulane Center for the Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribute.shtml). The human MSCs [12, 13] were isolated from 1–4-ml bone marrow aspirates taken from the iliac crest of normal adult donors after informed consent was obtained and under a protocol approved by an institutional review board. Nucleated cells were isolated with a density gradient (Ficoll-Paque; GE Healthcare, Piscataway, NJ, http://www.gehealthcare.com) and resuspended in complete culture medium consisting of 
-minimal essential medium (Gibco, Carlsbad, CA, http://www.invitrogen.com), 17% fetal bovine serum (FBS) lot selected for rapid growth of MSCs (Atlanta Biologicals, Inc., Norcross, GA, http://www.atlantabio.com), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Gibco). All of the nucleated cells (15–100 million) were plated in 20 ml of medium in a 175-cm2 culture dish (Nunc, Naperville, IL, http://www.nuncbrand.com) and incubated at 37°C with 5% CO2. After 24 hours, nonadherent cells were discarded, and adherent cells were thoroughly washed twice with phosphate-buffered saline (PBS). The cells were incubated 4–11 days until approximately 70% confluent, harvested with 0.25% trypsin and 1 mM EDTA for 5 minutes at 37°C, and replated at 50 cells per cm2 in an intercommunicating system of culture flasks (6,300 cm2; Cell Factory; Nunc). The cells were incubated 7–10 days until approximately 70% confluent, harvested with trypsin/EDTA, suspended at 1 x 106 cells per ml in 5% dimethyl sulfoxide and 30% FBS, and frozen in 1-ml aliquots at –80°C overnight and then placed in liquid nitrogen (passage 1 cells).
To expand the MSCs, a frozen vial was thawed, plated in a 57-cm2 culture dish, and incubated for 1 day, to recover viable adherent cells. MSCs were then replated at 50 cells per cm2 and incubated for 10 days without a change of medium in an intercommunicating system of culture flasks (Cell Factory; Nunc) or culture dishes (Nunc) at 37°C with 5% CO2 (passage 2 cells). Cell morphology was then observed, and pictures were taken over the next 10 days under phase-contrast microscopy. Each day, cells from three plates were harvested and counted with a hemacytometer.
Microarray Sample Preparation
Total RNA was extracted at passage 2 from day 2 and day 7 cultures from three donors of multipotent stromal cells using the RNAqueous Kit (Ambion, Austin, TX, http://www.ambion.com). Samples for microarrays were prepared according to the manufacturer's directions. In brief, 5 µg of total RNA was used to synthesize double-stranded cDNA (Superscript Choice System; Gibco). After synthesis, the double-stranded cDNA was purified by phenol/chloroform extraction (Phase Lock Gel; Eppendorf Scientific, Hamburg, Germany, http://www.eppendorf.com) and concentrated by ethanol precipitation. In vitro transcription was used to produce biotin-labeled cRNA (BioArray HighYield RNA Transcription Labeling Kit; Enzo Diagnostics, Farmingdale, NY, http://www.enzo.com). The biotinylated cRNA was then cleaned (RNeasy Mini Kit; Qiagen, Valencia, CA, http://www1.qiagen.com), fragmented, and hybridized on the HG-U133 Plus 2.0 microarray chips (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). The chips consisted of more than 54,000 transcripts, representing more than 31,000 human genes. After washing, individual microarray chips were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR, http://probes.invitrogen.com), amplified with biotinylated antistreptavidin (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and scanned for fluorescence (GeneChip Scanner 3000; Affymetrix) using GeneChip Operating software 1.0 (GCOS; Affymetrix).
Microarray Data Processing
GCOS recorded intensities for perfect match (PM) and mismatch (MM) oligonucleotides and determined whether genes were present, marginal, or absent. The scanned images were then transferred to the dChip program [14–16]. To allow comparisons of different microarrays, one array was chosen as the baseline array (220R-day 2, median intensity of 203) against which the other arrays were normalized at the probe intensity level. The dChip program then calculated the model based expression values using the PMs and MMs. Negative values were assigned a value of 1. Microarray data is available at the Gene Expression Omnibus database (GSE9520
[NCBI GEO]
).
Filtering of the Data
Data from six samples were analyzed (i.e., day 2 and day 7 from each of three donors of MSCs). To prepare for the clustering algorithm, the data were filtered with three different protocols: (a) no filtering of the transcripts; (b) filtering for genes with a present call in at least 25% of the samples and for genes with a value greater than 0.4 for the CV (SD of expression value for each gene divided by the mean expression value for that gene across all the samples); and (c) filtering by analysis of variance (ANOVA) for genes differentially expressed between day 2 and day 7 with a p value <.01 and retaining only the genes that were present in either all three day 2 or all three day 7 samples. The first filtering gave 54,675 transcripts, the second 4,245, and the third 2,909.
Hierarchical Clustering Algorithm in dChip
The dChip program was used to standardize the expression values for each gene by linearly adjusting their values across all samples to a mean of 0 with an SD of 1. Individual genes were then clustered using an algorithm in dChip program that determined the correlation coefficients (r values) for the normalized expression values (distances between genes were defined as 1 – r). Genes with the shortest distances between them were merged into supergenes, connected in a dendrogram by branches with lengths proportional to their genetic distances, and then merged (centroid linkage). This process was repeated n – 1 times until all genes had been clustered. A similar algorithm was also used to cluster the samples. The standardization and clustering methods followed published procedures [17, 18]. Transcripts obtained from the third filtering protocol were also subjected to gene clustering to generate a heat map.
Gene Ontology
The patterns from the heat map were studied for GeneOntology (GO) terms, to provide information on the cellular component, biological process, and molecular function of the gene products (http://www.geneontology.org) [19]. Removal of the redundant probe sets (based on Gene ID) reduced the number of genes for the ANOVA clustering from 2,909 to 2,286. Next, p values were calculated for each term using an exact hypergeometric distribution in the dChip program, to compare the frequencies of individual terms within the pattern to the frequencies of those terms on the entire microarray. p values <.01 were considered significant.
Comparisons
Genes that were upregulated on either day 2 or day 7 were identified with the dChip program. The criteria for upregulated genes were genes that were called present in the three samples from the different donors, whose upregulation was at least twofold, and whose fold change was within the 90% confidence boundary. The genes that were upregulated at day 2 (208) and at day 7 (264) in all three donors were studied further using GoSurfer (http://bioinformatics.bioen.uiuc.edu/gosurfer) [20]. Removal of the redundant probe sets (based on Gene ID) reduced the number of genes from 208 to 168 for genes upregulated at day 2 and from 264 to 193 for genes upregulated at day 7. The two groups (upregulated at day 2 and day 7) were tested for significance, highlighting the nodes with p value <.05 and hiding nodes with fewer than 30 genes. The genes upregulated on either day 2 or day 7 in any donor were also studied for pathways using GenMAPP 2.0 [21].
Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from passage 2 day 2 and day 7 cultures from three donors of MSCs using the RNAqueous Kit (Ambion), and 40 ng was converted into cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). A custom-designed TaqMan Low Density Array (Applied Biosystems) was used to perform real-time polymerase chain reaction (PCR). Samples with converted cDNA (40 ng in 50 µl) were adjusted with water to a final volume of 200 µl and mixed with an equal volume of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), and 10 ng of cDNA was transferred into each of four sample-loading ports on a microfluidics 384-well card preloaded with TaqMan Gene Expression Assay targets (Applied Biosystems) (64 targets with two endogenous controls in triplicate). Two samples of day 2 and day 7 cultures from each donor were assayed the card. Cards were analyzed with (ABI Prism 7900HT Sequence Detection System; Applied Biosystems) using the SDS 2.2 program (Applied Biosystems) with 18S as the endogenous control for normalization. Parameters used were as follows: automatic CT calculation, automatic outlier removal, and relative quantity (RQ) minimum/maximum confidence of 95.0%. When the baseline RQ minimum or maximum values did not overlap with the sample RQ values, the change in expression was considered significant.
Enzyme-Linked Immunosorbent Assay
Medium was harvested from passage 2 day 2 and day 7 cultures from three donors of multipotent stromal cells. Medium incubated without MSCs was used as a control. Samples were then filtered through 0.22-µm filters (Millipore, Billerica, MA, http://www.millipore.com) to remove any possible remaining cells. Enzyme-linked immunosorbent assays (ELISAs) were then performed using Quantikine Kits (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) according to the manufacturer's recommendations. In brief, samples and standards were added to microplates precoated with immobilized human monoclonal antibodies for leukemia inhibitory factor (LIF), interleukin 11 (IL-11), or pro-matrix metalloproteinase 1 (pro-MMP1). Then, an enzyme-linked monoclonal antibody against LIF, IL-11, or pro-MMP1 was added. Colorimetric substrate solution was added and developed, and absorbance intensity at 450 nm was measured.
Fluorescence-Activated Cell Scanning Analysis
MSCs were detached with EDTA/trypsin, suspended in Hanks' balanced saline solution, and assayed by FACScan analysis (Cytomics FC 500; Beckman Coulter, Miami, http://www.beckmancoulter.com). Antibodies against the following were used: CD36, CD34, CD45, CD11b, CD44, CD166, CD90, CD49b, CD105, CD117, CD59, CD79a (Beckman Coulter), CD147, CD49c, CD29, CD49f, HLA-abc, and HLA-class II (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com).
Approximately 200,000 cells were divided into aliquots in amber-tinted 1.5-ml centrifuge tubes, pelleted by centrifugation for 3 minutes at 1,020g, and resuspended. Various conjugated antibodies (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) were added to the cell suspension according to the manufacturer's specifications. The samples were incubated at room temperature in the dark for 20 minutes. The cells were pelleted, washed three times with PBS, resuspended in 0.3 ml of PBS, and assayed.
Cell Cycle Profile
Two hundred thousand MSCs in 100 µl of PBS were mixed with 100 µl of Reagent 1, RNase, and Triton X (DNA-Prep LPR; Beckman Coulter). The sample was gently vortexed for 30 seconds, and propidium iodide staining reagent (DNA-Prep Stain Reagent; Beckman Coulter) was added. After gentle vortexing for 5 seconds, the sample was incubated for 1 hour at room temperature in the dark. The sample was then assayed by flow cytometry, and cell cycle status was calculated by using ModFit LT software (Verity Software House, Topsham, ME, http://www.vsh.com).
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RESULTS
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Expansion of MSC Cultures from Three Donors
Frozen vials of passage 1 MSCs were thawed, plated at high density overnight to recover viable cells, replated at 50 cells per cm2, and incubated for up to 10 days. The cultures from all three donors expanded with characteristic lag, log, and near-stationary phases (Fig. 1). Cells from each donor also showed robust differentiation capacity in adipogenic and osteogenic assays (data not shown). The greatest fold change was observed on days 2 and 3 as the cells were about to exit from the lag phase (Fig. 1). The cells incubated for 2 days to approximately 5% confluence appeared small and spindle-shaped. Cells incubated for 7 days to approximately 70% confluence were larger and flatter. To define the changes in the cells, samples of day 2 and day 7 cultures were assayed by microarrays, real-time PCR, ELISAs, and FACScan analysis.
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Figure 1. Growth curves, photomicrographs, and schematic for the experiments. Passage 1 human multipotent stromal cells (MSCs) were plated at 50 cells per cm2 and incubated for up to 10 days without a change of medium. Assays in the schematic diagram were performed at days 2 and 7 for three donors, 220R (blue), 240L (red), and 5064L (black). Cells were counted and photomicrographs were taken every 24 hours for 10 days. Abbreviations: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; IL, interleukin; LIF, leukemia inhibitory factor; MMP, matrix metalloproteinase; PCR, polymerase chain reaction.
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Transcriptome Changes in MSCs During Expansion in Culture
RNA was isolated from passage 2 day 2 and day 7 cultures of three MSC donors and was used for microarray assays. Unbiased hierarchical clustering of the samples using all the genes on the microarrays (54,675) showed that the variations between donors were smaller than differences between days in culture (Fig. 2A). This observation was supported by using only genes that changed significantly (4,245) for unbiased sample clustering (Fig. 2B). To further investigate the differences between day 2 and day 7 samples, biased filtering was performed using ANOVA (2,909). Clustering of the samples (Fig. 2C) and genes (Fig. 2D) showed marked differences between day 2 and day 7 samples. Genes significantly upregulated at day 2 were primarily cell cycle-related, whereas protein transport- and metabolism-related genes were upregulated at day 7 (Fig. 2D).
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Figure 2. Microarray assays of the MSCs harvested on day 2 and day 7 from three donors. Microarray data were analyzed using dChip software. (A): Hierarchical clustering with no filtering of the transcripts. (B): Hierarchical clustering with filtering for genes with a present call in at least 25% of the samples and for genes with a value greater than 0.4 for the CV. (C, D): Hierarchical clustering with filtering by ANOVA for genes differentially expressed between day 2 and day 7 with a p value <.01 and retaining only the genes that were present in either all three day 2 or all three day 7 samples. The patterns from the heat map were then queried for GeneOntology term enrichment, with p values calculated using exact hypergeometric distribution. On the heat map, red indicates upregulation and blue indicates downregulation on the basis of genewise standardized values (scale bar on the bottom). (E): Venn diagrams of genes listed were present and upregulated at least twofold with a 90% confidence interval. There were 208 upregulated genes at day 2 and 264 at day 7 in common among the three donors. Abbreviation: ANOVA, analysis of variance.
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To study the differences between day 2 and day 7 cultures from each donor, the genes that were called present and changed at least twofold in each donor were examined. This resulted in 385–1,390 genes that were upregulated at day 2, whereas 636–1,680 genes were upregulated at day 7. There were 208 upregulated genes at day 2 that were shared among all three donors, whereas 264 genes were upregulated in all three donors at day 7 (Fig. 2E). Genes that were upregulated significantly in all donors at day 2 were involved in cell division, whereas developmental genes were upregulated at day 7 (supplemental online Table 3; supplemental online Fig. 1). Fold changes for genes from selected GO categories are shown in Table 1(upregulated at day 2) and Table 2(upregulated at day 7). Both day 2 and day 7 sample groups had several transcription factors and receptors upregulated. Cell surface receptors integrin 6 and 2 were upregulated at day 2, whereas integrin 7 and 11 were upregulated at day 7 (Tables 1 and 2). Cell cycle genes were upregulated in day 2 samples, including proliferation markers cyclin A2, chromatin assembly factor 1, and Ki-67 antigen (Table 1). Extracellular matrix genes were upregulated in day 7 samples, including lumican, decorin, and elastin (Table 2).
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Table 1. Mean fold changes (lower bound, 90% confidence) of genes that were differentially expressed (upregulated) in all three donors after 2 days of culture compared with 7 days in culture (selected GeneOntology groups shown)
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Table 2. Mean fold changes (lower bound, 90% confidence) of genes that were differentially expressed (upregulated) in all three donors after 7 days of culture compared with 2 days in culture (selected GeneOntology groups shown)
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To investigate the biological pathways involved in MSC expansion, genes that were either upregulated in day 2 or day 7 in at least one of the three donors were analyzed using GenMAPP software. Positive regulators of proliferation were shown to be upregulated at day 2, whereas inhibitors of proliferation were upregulated at day 7 (supplemental online Fig. 2).
Confirmation of Microarray Results Using Real-Time PCR and ELISA
To confirm some of the microarray data, real-time PCR assays were performed using low-density arrays customized for 64 target genes in triplicate. There were similar increases and decreases for the genes in assays by both real-time PCR and microarrays (Fig. 3). In general, the fold changes detected by real-time PCR were larger than those in the microarrays. Real-time PCR assays were also more sensitive in that changes of 11 genes in donor 5064L and of 11 genes in donor 240L were statistically significant in real-time PCR but not significant in microarrays (Fig. 3). Based on real-time PCR, the genes with the highest fold changes at day 2 were MMP1, PODXL, DKK1, and AURKB for donor 5064L and ITGA2, MMP1, PODXL, and IL-11 for donor 240L. Genes with the highest fold changes at day 7 were SYNPO2, AGC1, THBS2, and FGF7 for donor 5064L and SYNPO2, AGC1, ITGA7, and GDF5 for donor 240L (Fig. 3).
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Figure 3. Real-time PCR assays. In most cases, the magnitude of fold change was greater in real-time PCR, but all genes changed with the same directionality. Abbreviations: AGC, aggrecan; ANGPT, angiopoietin; AURKB, aurora kinase B; CCND, cyclin D; CDKN, cyclin-dependent kinase inhibitor; CTNNB, catenin beta; DCN, decorin; DKK, dickkopf homolog; E2F7, E2F transcription factor 7; EPHB, ephrin receptor B; FGF, fibroblast growth factor; FZD, frizzled homolog; GAS, growth arrest-specific; GDF, growth differentiation factor; GPC, glypican; GSN, gelsolin; HMMR, hyaluronan-mediated motility receptor; HIF1A, hypoxia-inducible factor 1 alpha; IL, interleukin; ITGA, integrin alpha; LIF, leukemia inhibitory factor; LPXN, leupaxin; LUM, lumican; MCM, minichromosome maintenance deficient; MK167, antigen identified by monoclonal antibody Ki-67; MMP, matrix metalloproteinase; NOTCH, notch homolog; PCR, polymerase chain reaction; PODXL, podocalyxin-like; RARB, retinoic acid receptor beta; RUNX, runt-related transcription factor; SDC, syndecan; SOCS, suppressor of cytokine signaling; SOX, sex determining region Y-box; STAT, signal transducer and activator of transcription; SYNPO, synaptopoctin; SYTL, synaptotagmin-like; THBS, thrombospondin; VCAM, vascular cell adhesion molecule; VIL, villin; WNT, wingless-type MMTV integration site; WWTR, ww domain containing transcription regulator.
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The production of secreted factors selected from microarray/real-time PCR data was measured by ELISAs. The concentration of secreted IL-11, LIF, and MMP1 was measured from medium conditioned for 0, 2, or 7 days. MSCs from all donors produced detectable amounts of IL-11 by day 2 with increasing amounts at day 7. LIF production was detectable at day 7 in all donors. Secreted MMP1 could be detected at day 7 in donors 240L and 5064L (Fig. 4).
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Figure 4. Enzyme-linked immunosorbent assays demonstrated the presence of secreted factors in MSC-conditioned medium. Secreted factors (IL-11, LIF, and MMP1) identified in microarray/real-time polymerase chain reaction data were present at higher levels in Day 7 conditioned medium. IL-11 was present as early as Day 2. However, donor 220R showed no MMP1 present in this assay. Abbreviations: CCM, complete culture medium; IL, interleukin; LIF, leukemia inhibitory factor; MMP, matrix metalloproteinase.
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Analysis of Cell Cycle Profiles and Surface Epitopes
To confirm proliferation-related observations, an analysis of cell cycle status was performed at day 2 and day 7 using a FACScan. Nearly 50% of the cells at day 2 were in S-phase and actively dividing, whereas less than 10% of the cells at day 7 were in S-phase. MSCs were largely quiescent at day 7, where they were stalled at G1 (Fig. 5). FACScan analysis showed characteristic MSC surface epitopes present, consistent with microarray and real-time PCR results (supplemental online Figs. 3, 4; supplemental online Table 3).
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Figure 5. Cell cycle analyses. MSCs at day 2 and day 7 were analyzed for propidium iodide incorporation by FACScan analysis. MSCs from donors 220R, 240L, and 5064L had 45%, 43%, and 46%, respectively of cells at S-phase at day 2 and 10%, 5%, and 7% at day 7. Abbreviation: FL, fluorescence.
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DISCUSSION
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The experiments here were designed to define the changes in multipotent stromal cells as they are expanded in culture. On day 2 after plating at low density, early-passage MSCs are in transition from a lag phase to log phase. On day 7, the cells are in a stationary phase. Therefore, MSCs from day 2 and day 7 cultures should reflect the major changes as the cells are expanded.
Data from MSCs from three different donors of bone marrow aspirates demonstrated marked differences in patterns of gene expression on day 2 and day 7. However, the variation among three donors was less than the variation with the expansion of MSCs from a single donor. As expected, the genes upregulated on day 2 were primarily genes for cell division, whereas the genes upregulated on day 7 were primarily genes for development, morphogenesis, and physiological processes. Forty-two genes involved in cell cycle were upregulated on day 2 but only 8 on day 7. In contrast, 16 genes involved in extracellular matrix were upregulated on day 7 and only 4 on day 2.
The microarray data for the expression patterns of more than 50 of the genes were verified by real-time PCR assays. Increases and decreases similar to those of the microarray assays were observed, but the fold changes for several genes were larger. The expression patterns of several other genes were verified by both ELISA and FACScan analysis. Among the significant changes detected by real-time PCR were upregulation of DKK1 and PODXL on day 2. Expression of DKK1, an inhibitor of the Wnt signaling pathway, was previously shown to be required for reentry of MSCs into cell cycling [22]. PODXL is a member of the CD34 family of sialomucins that are characteristically expressed on progenitor cells [23]. Two of the genes upregulated on day 2 were previously found to be expressed in other stem cells [24, 25]: integrin 6 and ephrin B2 (Table 1; Fig. 3). Therefore, the results are consistent with previous indications that low-density cultures of MSCs are enriched for early progenitor cells that replicate more rapidly and that are more efficient both in osteogenic and adipogenic differentiation [12, 13] and in engraftment in vivo [26]. Chondrogenic differentiation was more efficient with day 7 cultures than with day 4 cultures that were initially plated at 50 cells per cm2 [12], an observation that may be complicated by the need to transfer the cells to serum-free medium and prepare them as micromass cultures to assay chondrogenesis.
MSCs from bone marrow have been used extensively to examine cellular differentiation in culture and in vivo and for their therapeutic potential both in animal models for diseases and in patients. However, many of the observations are difficult to reconcile. For example, there is no general agreement whether MSCs repair injured tissues by differentiating to replace damaged cells, by cell fusion, by secreting cytokines and chemokines that suppress apoptosis or that stimulate expansion of tissue-endogenous stem/progenitor cells, by suppressing inflammatory and immune reactions, or perhaps by transfer on mitochondria [7, 27–31]. Differences in the properties of different preparations of MSCs could well explain discrepancies in data reported by different laboratories [32]. The results presented here raise the possibility that the effectiveness of MSCs in enhancing repair of tissues by one or more of these processes may differ depending on the precise protocols used to isolate and expand the cells.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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Supported in part by NIH Grants HL073755, HL073252, and P40 RR 17447; by HCA; the W.M. Keck Foundation; and the Louisiana Gene Therapy Research Consortium. B.L.L. and J.Y. contributed equally to this work.
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Abstract
Introduction