Gene Expression Profiles Of Mesenchymal Stem Cells Research Articles

Implication of therapeutic outcomes associated with molecular characterization of paediatric aplastic anaemia

ObjectivesSevere aplastic anemia is characterized by a hypocellular bone marrow and peripheral cytopenia. Mesenchymal stem cells (MSCs) play a crucial role in haematopoietic stem cells (HSCs) development and the development of microenvironment suitable for hematopoiesis. Molecular characterization of telomere maintenance pathway and gene expression profiling of MSCs can be important for the therapeutic interventions among paediatric aplastic anaemia patients. MethodsThe study involved paediatric aplastic anaemia patients (n = 10) and age matched paediatric healthy donors (n = 8). Peripheral blood samples were collected from the individuals. Average leucocyte telomere length and gene expression of the telomere maintenance genes were determined by quantitative real time PCR. Microarray based gene expression profiles (GSE33812) of MSCs for five paediatric aplastic anaemia patients were analyzed compared to five healthy controls and the data was downloaded from the GEO database. ResultsThe telomere length was significantly shorter among paediatric AA patients compared to age matched healthy donors. Interestingly, one subgroup (n = 2) of paediatric AA patients has moderate telomere length comparable to age matched healthy donors. Based on the gene expression analysis of telomere maintenance pathway, TERF2 was significantly downregulated among paediatric patients with shorter telomere length but not among paediatric patients with moderate telomere length. Gene expression profiling of MSCs revealed three differentially expressed genes (GAS2L3, MK167 and TMSB15A) among the patients and was associated with therapeutic outcome. ConclusionTelomere length estimation and gene expression patterns of the MSCs and telomere length maintenance pathway may serve as a potential biomarker and could be associated with therapeutic choice of paediatric aplastic anaemia patients.

Comparative transcriptomic analysis of human mesenchymal stem cells derived from dental pulp and adipose tissues

Objective: Mesenchymal stem cells (MSCs) have been isolated from various human tissues. Although they share cardinal stem cell features of self-renewal and multi-potency, they also seem to possess distinct characteristics depending on the tissue types they originated from. When developing stem cell-based therapies, MSCs with the most desirable characteristics should be chosen. However, our knowledge on tissue type-specific characteristics of MSCs is limited. Here, we comparatively studied the gene expression profiles of MSCs from different tissue types, and predicted target diseases suitable for each type of MSCs. Methods: We harvested MSCs from human dental pulp and adipose tissue specimens and subjected them to gene expression microarray analysis. Characteristic gene expression signatures of the MSCs from each tissue type were identified using gene-annotation enrichment analysis. Results: Dental pulp-derived MSCs exhibited gene expression signatures of neuronal growth, while adipose tissue-derived MSCs exhibited signatures of angiogenesis and hair growth. MSCs from each tissue type expressed a discrete set of genes encoding secretory peptides, which may function as paracrine factors. Conclusions: MSCs derived from different tissue types demonstrated distinct gene expression signatures, which are suggestive of target diseases in clinical applications of the MSCs and stem cell-conditioned media. By expanding the analysis to MSCs from a wide range of tissue types, and by employing multiple omics approaches, a catalogue of MSCs and therapeutic targets can be generated.

Effect of cell source and osteoblast differentiation on gene expression profiles of mesenchymal stem cells derived from bone marrow or adipose tissue.

Mesenchymal stem cells (MSCs) have been used in therapies for bone tissue healing. The aim of this study was to investigate the effect of cell source and osteoblast differentiation on gene expression profiles of MSCs from bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) to contribute for selecting a suitable cell population to be used in cell-based strategies for bone regeneration. BM-MSCs and AT-MSCs were cultured in growth medium to keep MSCs characteristics or in osteogenic medium to induce osteoblast differentiation (BM-OBs and AT-OBs). The transcriptomic analysis was performed by microarray covering the entire rat functional genome. It was observed that cells from bone marrow presented higher expression of genes related to osteogenesis, whereas cells from adipose tissue showed a higher expression of genes related to angiogenesis and adipocyte differentiation, irrespective of cell differentiation. By comparing cells from the same source, MSCs from both sources exhibited higher expression of genes involved in angiogenesis, osteoblast differentiation, and bone morphogenesis than osteoblasts. The clustering analysis showed that AT-OBs exhibited a gene expression profile closer to MSCs from both sources than BM-OBs, suggesting that BM-OBs were in a more advanced stage of differentiation. In conclusion, our results suggest that in cell-based therapies for bone regeneration AT-MSCs could be considered for angiogenic purposes, whereas BM-MSCs and osteoblasts differentiated from either source could be better for osteogenic approaches.

Soft Elasticity-Associated Signaling and Bone Morphogenic Protein 2 Are Key Regulators of Mesenchymal Stem Cell Spheroidal Aggregates.

Cell therapy with adult mesenchymal stem cells (MSCs) is a promising approach to regenerative medicine and autoimmune diseases. There are various approaches to improve the efficacy of MSC-based therapeutics, and MSC preparation as spheroidal aggregates, or MSC spheroids, is a novel preparatory and delivery method. Spheroid formation induces a dramatic change in the gene expression profile of MSCs. Self-activation of interleukin-1 (IL1) signaling was shown to be upstream of both pro- and anti-inflammatory genes in MSC spheroids, but the molecular pathways that initiate IL1 signaling remain unknown. As bone morphogenic protein (BMP)2 upregulation precedes that of IL1B expression during spheroid formation, we hypothesized that BMP2 signaling triggers IL1 signaling in MSC spheroids. Contrary to expectations, BMP2 signaling decreased expression of IL1B and downstream genes in a SMAD6-dependent manner. Conversely, IL1B signaling enhanced BMP2 expression. Another major difference between two-dimensional (2D) monolayer culture and three-dimensional (3D) spheroid culture is the Young's elasticity modulus, or stiffness, of the materials surrounding the cells, as there is a million-fold difference between a plastic surface for standard 2D culture (GPa) and 3D spheroidal aggregates (0.1 kPa). We tested another hypothesis that soft elasticity-associated mechano-signaling initiates the gene expression change during spheroid formation. Results showed that both BMP2 expression and inflammatory signaling are upregulated in an elasticity-associated signaling-dependent manner in MSCs. Lastly, BMP2 signaling enhanced cell survival and cell spreading of MSC spheroids. In summary, our study suggests that soft elasticity and BMP2 signaling are critical for MSC spheroids.

Poly-L-lysine Prevents Senescence and Augments Growth in Culturing Mesenchymal Stem Cells Ex Vivo.

Mesenchymal stem cells (MSCs) possess great therapeutic potential. Efficient in vitro expansion of MSCs is however necessary for their clinical application. The extracellular matrix (ECM) provides structural and biochemical support to the surrounding cells, and it has been used as a coating substrate for cell culture. In this study, we have aimed to improve the functionality and stemness of MSCs during culture using poly-L-lysine (PLL). Functionality of MSCs was analysed by cell cycle analysis, differentiation assay, β-galactosidase staining, and RT-PCR. Furthermore, we assessed the global gene expression profile of MSCs on uncoated and PLL-coated plates. MSCs on PLL-coated plates exhibited a faster growth rate with increased S-phase and upregulated expression of the stemness markers. In addition, their osteogenic differentiation potential was increased, and genes involved in cell adhesion, FGF-2 signalling, cell cycle, stemness, cell differentiation, and cell proliferation were upregulated, compared to that of the MSCs cultured on uncoated plates. We also confirmed that MSCs on uncoated plates expressed higher β-galactosidase than the MSCs on PLL-coated plates. We demonstrate that PLL provides favourable microenvironment for MSC culture by reversing the replicative senescence. This method will significantly contribute to effective preparation of MSCs for cellular therapy.

Spheroid Culture of Mesenchymal Stem Cells

Compared with traditional 2D adherent cell culture, 3D spheroidal cell aggregates, or spheroids, are regarded as more physiological, and this technique has been exploited in the field of oncology, stem cell biology, and tissue engineering. Mesenchymal stem cells (MSCs) cultured in spheroids have enhanced anti-inflammatory, angiogenic, and tissue reparative/regenerative effects with improved cell survival after transplantation. Cytoskeletal reorganization and drastic changes in cell morphology in MSC spheroids indicate a major difference in mechanophysical properties compared with 2D culture. Enhanced multidifferentiation potential, upregulated expression of pluripotency marker genes, and delayed replicative senescence indicate enhanced stemness in MSC spheroids. Furthermore, spheroid formation causes drastic changes in the gene expression profile of MSC in microarray analyses. In spite of these significant changes, underlying molecular mechanisms and signaling pathways triggering and sustaining these changes are largely unknown.

Benefits of biphasic calcium phosphate hybrid scaffold-driven osteogenic differentiation of mesenchymal stem cells through upregulated leptin receptor expression.

BackgroundThe use of mesenchymal stem cells (MSCs) and coralline hydroxyapatite (HA) or biphasic calcium phosphate (BCP) as a bone substitute for posterolateral spinal fusion has been reported. However, the genes and molecular signals by which MSCs interact with their surrounding environment require further elucidation.MethodsMSCs were harvested from bone grafting patients and identified by flow cytometry. A composite scaffold was developed using poly(lactide-co-glycolide) (PLGA) copolymer, coralline HA, BCP, and collagen as a carrier matrix for MSCs. The gene expression profiles of MSCs cultured in the scaffolds were measured by microarrays. The alkaline phosphatase (ALP) activity of the MSCs was assessed, and the expression of osteogenic genes and proteins was determined by quantitative polymerase chain reaction (Q-PCR) and Western blotting. Furthermore, we cultured rabbit MSCs in BCP or coralline HA hybrid scaffolds and transplanted these mixtures into rabbits for spinal fusion. We investigated the differences between BCP and coralline HA hybrid scaffolds by dual-energy X-ray absorptiometry (DEXA) and computed tomography (CT).ResultsTested in vitro, the cells were negative for hematopoietic cell markers and positive for MSC markers. There was higher expression of 80 genes and lower of 101 genes of MSCs cultured in BCP hybrid scaffolds. Some of these genes have been shown to play a role in osteogenesis of MSCs. In addition, MSCs cultured in BCP hybrid scaffolds produced more messenger RNA (mRNA) for osteopontin, osteocalcin, Runx2, and leptin receptor (leptin-R) than those cultured in coralline HA hybrid scaffolds. Western blotting showed more Runx2 and leptin-R protein expression in BCP hybrid scaffolds. For in vivo results, 3D reconstructed CT images showed continuous bone bridges and fusion mass incorporated with the transverse processes. Bone mineral content (BMC) values were higher in the BCP hybrid scaffold group than in the coralline HA hybrid scaffold group.ConclusionsThe BCP hybrid scaffold for osteogenesis of MSCs is better than the coralline HA hybrid scaffold by upregulating expression of leptin-R. This was consistent with in vivo data, which indicated that BCP hybrid scaffolds induced more bone formation in a spinal fusion model.

The Effect of Hypoxia on Mesenchymal Stem Cell Biology.

Although physiological and pathological role of hypoxia have been appreciated in mammalians for decades however the cellular biology of hypoxia more clarified in the past 20 years. Discovery of the transcription factor hypoxia-inducible factor (HIF)-1, in the 1990s opened a new window to investigate the mechanisms behind hypoxia. In different cellular contexts HIF-1 activation show variable results by impacting various aspects of cell biology such as cell cycle, apoptosis, differentiation and etc. Mesenchymal stem cells (MSC) are unique cells which take important role in tissue regeneration. They are characterized by self-renewal capacity, multilineage potential, and immunosuppressive property. Like so many kind of cells, hypoxia induces different responses in MSCs by HIF- 1 activation. The activation of this molecule changes the growth, multiplication, differentiation and gene expression profile of MSCs in their niche by a complex of signals. This article briefly discusses the most important effects of hypoxia in growth kinetics, signalling pathways, cytokine secretion profile and expression of chemokine receptors in different conditions.

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.

Gene expression analysis of laminin-1-induced neurite outgrowth in human mesenchymal stem cells derived from bone marrow.

The mechanisms underlying the differentiation of Mesenchymal stem cells (MSCs) toward neuronal cell type are not clearly understood. Earlier, we reported that laminin-1 induces neurite outgrowth in human MSCs via c-Jun/AP-1 activation through ERK, JNK, and Akt pathways. In this study, we demonstrate that laminin-1 increases the expression of proneural gene, neuroD1 and induces the expression of immediate-early biomarkers of neuronal cell-programming-Egr1, Egr3, PC3, and PC4. Gene expression profiling of MSCs cultured on laminin-1 and Poly-l-lysine for 12 h revealed differential regulation of 267 genes (>1.5 fold, p < 0.05), predominantly in the category of nervous system development and affected the pathways involved in TGF-β/TNF-α signaling, regulation of MAPK and JNK cascade. Data for 11 selected genes related to nervous system development was validated by real time PCR. Transcriptional regulatory network analysis revealed c-Jun as the key transcription factor regulating majority of differentially expressed genes and identified Disrupted in schizophrenia 1, as a novel target of c-Jun. Modeling and analysis of biological network showed selective induction of Growth Arrest and DNA damage 45 (GADD45B) and repression of NF-κB inhibitor A (NFκBIA). Collectively, our findings provide the basis for understanding the molecular mechanisms associated with laminin-1-induced neurogenic expression in MSCs.

Study of differential effects of TGF-beta3/BMP2 on chondrogenesis in MSC cells by gene microarray data analysis

In order to explore the differential effects of TGF-beta3 and BMP2 on chondrogenesis in mesenchymal stem cells (MSCs), the gene expression profiles of MSC treated with TGF-beta3 and BMP2 were subjected to systematic analysis on the gene and functional level. The gene expression profiles of MSCs (downloaded from Gene Expression Omnibus database) in the early and later stages, induced with TGF-beta2 and BMP2, were analyzed using packages within R software and the differentially expressed genes (DEGs) were screened. The DEGs both in the two experimental groups were subjected to Gene Ontology and pathway enrichment analysis. The protein-protein interaction (PPI) networks of the DEGs were constructed using cytoscape software. Among the DEGs, 1,194 genes were up-regulated and 580 genes were down-regulated. The up-regulated genes were mainly enriched in the TGF-beta and cell cycle signaling pathways and down-regulated genes were mainly enriched in the insulin-mediated signal pathway, metabolic pathway of fructose and mannose, and glycolysis/gluconeogenesis pathway. Based on the PPI network analysis, the genes of KIAA0101, NEDD4, and TINF2 were confirmed to be important on chondrogenesis. The analysis of DEGs both in TGF-beta3 and BMP2 treated MSCs indicates that the genes are mainly involved in the cell cycle and intracellular signaling pathways. Also the similar gene expression profile can be achieved by transcription factors or microRNAs (miR-199a-5p and miR-31-5p) based on our prediction, which can provide a new approach for the treatment of cartilage injury.

Gene expression profile study on osteoinductive effect of natural hydroxyapatite

The aim of this study was to investigate the osteoinductive effect of natural hydroxyapatite (NHA). NHA was extracted from pig bones and prepared into disk-like samples. Then, proliferation of mouse bone mesenchymal stem cells (MSCs) cultured on NHA was assessed by the methylthiazoltetrazolium (MTT) assay. Furthermore, microarray technology was applied to obtain the gene expression profiles of MSCs cultured on NHA at 24, 48, and 72 h. The gene expression profile was then comprehensively analyzed by clustering, Gene Ontology (GO), Gene Microarray Pathway Profiler (GenMAPP) and Ingenuity Pathway Analysis (IPA). According to the results of microarray experiment, 8992 differentially expressed genes were obtained. 90 differential expressed genes related to HA osteogenic differentiation were determined by GO analysis. These genes included not only 6 genes related to HA osteogenic differentiation as mentioned in the literatures but also newly discovered 84 genes. Some important signaling pathways (TGF-β, MAPK, Wnt, etc.) were influenced by these genes. Gene interaction networks were obtained by IPA software, in which the scoring values of two networks were highest, and their main functions were related to cell development. The comprehensive analysis of these results indicate that NHA regulate some crucial genes (e.g., Bmp2, Spp1) and then activate some pathways such as TGF-β signaling pathway, and ultimately osteogenic differentiation was induced.

Migration potential and gene expression profile of human mesenchymal stem cells induced by CCL25

Recruitment of mesenchymal stem cells (MSC) to tissue damages is a promising approach for in situ tissue regeneration. The physiological mechanisms and regulatory processes of MSC trafficking to injured tissue remain poorly understood. However, the pivotal role of chemokines in MSC recruitment has already been shown. The aim of this study was to determine the migratory potential and the gene expression profile of MSC stimulated with the CC chemokine CCL25 (TECK). Bone marrow derived human MSC were exposed to different doses of CCL25 in a standardized chemotaxis assay. Microarray gene expression profiling and pathway analysis were performed for CCL25 stimulated MSC. Maximum migration of MSC towards CCL25 was observed at 10 3 nM. Microarray analysis revealed an induction of molecules directly involved in chemotaxis and homing of bone marrow cells (CXCL1–3, CXCL8, PDE4B), cytoskeletal and membrane reorganisation (CXCL8, PLD1, IGFBP1), cellular polarity (PLD1), and cell movement (CXCL1–3, CXCL6, CXCL8, PTGS2, PDE4B, TGM2). Respective chemokine secretion was confirmed by protein membrane-array analysis. The activation of CXCR2 ligands (CXCL1–3, CXCL5–6, CXCL8) and a LIF-receptor/gp130 ligand (LIF) indicated an involvement of the respective signaling pathways during initiation of chemotaxis and migration. These results suggest CCL25 as a new potential candidate for further in situ regeneration approaches.

Effects of gelatin on proliferation of cord blood derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are isolated from various tissues including bone marrow (BM), placenta, adipose tissue, and umbilical cord blood (CB). Compared to MSC isolated from BM (BM‐MSC), MSC from cord blood (CB‐MSC) has a short logarithmic growth stage before entering the senescence stage in vitro long‐term culture. Gelatin coated dishes are used to culture for embryonic stem cells, primary cell and clones. To determine whether the usage of gelatin‐coated dishes gave superior effects on efficient isolation MSC from CB and longer proliferation of CB‐MSC in vitro, we cultured mononuclear cells (MNC) isolated from human CB with gelatin‐coated or uncoated dishes. In the results, CB‐MSC in gelatin‐coated dishes revealed higher and longer proliferation than those in uncoated dishes. To unveil the mechanism, we compared gene expression profiles of MSC cultured in gelatin‐coated and uncoated dishes using the microarray technology. Many of genes involved in adhesion molecule induced signaling pathway were identified. Our findings suggest that gelatin‐derived signaling may contribute to the expended proliferation and maintenance of CB‐MSC and confer the chance to study what signals involved to CB‐MSC proliferation.

Prospective Identification and Characterization of Human Mesenchymal Stem Cell Subpopulations.

Mesenchymal stem cells (MSC) are multipotent cells that readily differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes. They support hematopoiesis (stroma function) and have potent immunomodulatory properties making them promising candidates for clinical use. Although MSC cultures are heterogeneous in morphology, no specific markers are available that would allow for the prospective isolation of distinct subpopulations. Therefore we investigated whether potential MSC subpopulation markers could be identified by gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics). Human MSC were generated in standard cultures and stained with carboxyfluoresceine-succinimidylesther (CFSE) for cell division tracking. FACS analysis of CFSE-stained cells after 10 days culture showed that the majority of cells had undergone more than 3 cell divisions (51.2 ± 1.7%) whereas only 3.5 ± 0.9 % had not divided. Based on these results MSC were sorted by FACS according to their divisional history into non-proliferating cells (NP-MSC) and rapidly-proliferating cells (RP-MSC). Interestingly, the CFU-F frequencies of the NP-MSC were considerably (5-times) lower compared to RP-MSC. Furthermore, RP-MSC with low forward-/side-scatter properties had a higher CFU-F frequency than those with high scatter properties. Comparative oligonucleotide microarray analysis of FACS-sorted NP-MSC and RP-MSC populations identified a total of 112 differentially (≥4-fold) expressed genes. Fifty genes were significantly higher expressed in RP-MSC with 12 of them being cell cycle associated. Sixty-two genes showed a higher expression in NP-MSC and more than 20 of them were related to adhesion molecules, extracellular matrix proteins or cell senescence. Two of the differentially expressed genes (VCAM-1, FMOD) that corresponded to cell surface molecules were further tested for their potential to prospectively identify MSC subpopulations. Remarkably, FACS analysis using anti-VCAM-1 (CD106) and anti-fibromodulin (fmod) antibodies showed that approximately 40% and 3% of MSC were CD106neg/fmodneg and CD106pos/fmodpos, respectively. MSC were then sorted by FACS according to their expression of these markers and assayed for CFU-F frequency. Significant differences in CFU-F frequencies were observed between the double-negative and the double-positive MSC (12.3 ± 2.8 and 4.7 ± 2.1 colonies per 100 cells for CD106neg/fmodneg and CD106pos/fmodpos MSC, respectively). Experiments to test for differentiation potential differences are ongoing. Taken together, the data clearly demonstrate functional differences between MSC subpopulations. Furthermore, cell sorting based on proliferation characteristics and gene expression profiling enabled to identify surface markers that allowed for the first time to prospectively identify MSC subpopulations.

Differential Gene Expression Associated with Migration of Mesenchymal Stem Cells to Conditioned Medium from Tumor Cells or Bone Marrow Cells

Distinct signals that guide migration of mesenchymal stem cells (MSCs) to specific in vivo targets remain unknown. We have used rat MSCs to investigate the molecular mechanisms involved in such migration. Rat MSCs were shown to migrate to tumor microenvironment in vivo, and an in vitro migration assay was used under defined conditions to permit further mechanistic investigations. We hypothesized that distinct molecular signals are involved in the homing of MSCs to tumor sites and bone marrow. To test this hypothesis, gene expression profiles of MSCs exposed in vitro to conditioned medium (CM) from either tumor cells or bone marrow were compared. Analysis of the microarray gene expression data revealed that 104 transcripts were upregulated in rat MSCs exposed to CM from C85 human colorectal cancer cells for 24 hours versus control medium. A subset of 12 transcripts were found to be upregulated in rat MSCs that were exposed to tumor cell CM but downregulated when MSCs were exposed to bone marrow CM and included CXCL-12 (stromal cell-derived factor-1 [SDF-1]), CXCL-2, CINC-2, endothelial cell specific molecule-1, fibroblast growth factor-7, nuclear factor-kappaB p105, and thrombomodulin. Exposure to tumor cell CM enhanced migration of MSCs and correlated with increased SDF-1 protein production. Moreover, knockdown of SDF-1 expression in MSCs inhibited migration of these cells to CM from tumor cells, but not bone marrow cells, confirming the importance of SDF-1 expression by MSCs in this differential migration. These results suggest that increased SDF-1 production by MSCs acts in an autocrine manner and is required for migratory responses to tumor cells.

Mesenchymal Stem Cells, Fibroblasts and Pericytes: Different Functional States of the Same Cell?.

Mesenchymal stem cells (MSC) are multipotent precursors present in the bone marrow (BM), capable of differentiating into osteoblasts, adipocytes, chondrocytes and myoblasts. In addition to BM, MSC can be obtained from a variety of adult and fetal tissues. Additionally pericytes, that form a continuous subendothelial network of the vascular bed, share many properties with MSC, whereas the name stromal cell or fibroblast is often used interchangeably with MSC. How similar are MSCs obtained from different human tissues and what is their relationship with fibroblasts and pericytes? To answer these questions we have examined the expression of a set of 30 genes in 18 different human cell lines cultured in vitro: 7 MSC cultures from different sources (3 adult tissues: bone marrow, saphena vein, adipose tissue, and 4 fetal tissues: artery, liver, umbilical artery and umbilical vein), 2 of fibroblasts, 2 MSC cultures differentiated in vitro into adipocytes or osteoblasts, and one each of pericytes from the human retina, of bone marrow, endothelial cells, liver, brain, skeletal muscle and heart. Genes were selected on the basis of previous results of SAGE for MSC, CD34+ cells and fibroblasts. Cluster analysis using the software Cluster 3.0 UPGMA (average linkage) showed that all the MSC lines formed a very close cluster with pericytes and fibroblasts, separated from other normal human cells. Changes of the expression of highly expressed MSC genes were modest during in vitro differentiation of MSCs into osteoblasts or adipocytes, although the differentiation was accompanied by the increased expression of tissue specific genes. In addition, all the MSC lines had similar immunophenotypic markers and capacity for in vitro differentiation. Similarity of the gene expression profiles of MSC and fibrobasts were further confirmed by clustering of the 1,000 top expressed tags of SAGE libraries for 21 normal human tissues. Despite the similarity, expression of genes related to angiogenesis, especially CXCL6, was higher in MSC from umbilical vein, adult saphena vein and in pericytes than in BM-derived MSC. In conclusion, MSC that can be obtained from a variety of adult and fetal tissues have very similar biological markers, differentiation potential and gene expression profiles. Comparison of these characteristics also shows that MSC, fibroblasts and pericytes are very closely related, representing probably different functional states of the same cell. Finally, differentiaton of MSC into osteoblasts or adipocytes is marked by the expression of tissue specific genes, without dramatical changes of the overall profile.