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1.  Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential 
Introduction
Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.
Methods
The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA.
Results
The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied.
Conclusions
The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes.
doi:10.1186/scrt414
PMCID: PMC4055040  PMID: 24559797
2.  Hypoxic Preconditioning Results in Increased Motility and Improved Therapeutic Potential of Human Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(8):2173-2182.
Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential.
doi:10.1634/stemcells.2007-1104
PMCID: PMC3017477  PMID: 18511601
Immune-deficient mice; Human stem cells; Mesenchymal stem cells; Hypoxia; Transplantation; Tissue repair
3.  Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner 
PLoS ONE  2012;7(9):e46483.
Background
Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.
Methodology/Principal Findings
Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.
Conclusions/Significance
Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.
doi:10.1371/journal.pone.0046483
PMCID: PMC3459928  PMID: 23029528
4.  In vitro biosafety profile evaluation of multipotent mesenchymal stem cells derived from the bone marrow of sarcoma patients 
Background
In osteosarcoma (OS) and most Ewing sarcoma (EWS) patients, the primary tumor originates in the bone. Although tumor resection surgery is commonly used to treat these diseases, it frequently leaves massive bone defects that are particularly difficult to be treated. Due to the therapeutic potential of mesenchymal stem cells (MSCs), OS and EWS patients could benefit from an autologous MSCs-based bone reconstruction. However, safety concerns regarding the in vitro expansion of bone marrow-derived MSCs have been raised. To investigate the possible oncogenic potential of MSCs from OS or EWS patients (MSC-SAR) after expansion, this study focused on a biosafety assessment of MSC-SAR obtained after short- and long-term cultivation compared with MSCs from healthy donors (MSC-CTRL).
Methods
We initially characterized the morphology, immunophenotype, and differentiation multipotency of isolated MSC-SAR. MSC-SAR and MSC-CTRL were subsequently expanded under identical culture conditions. Cells at the early (P3/P4) and late (P10) passages were collected for the in vitro analyses including: sequencing of genes frequently mutated in OS and EWS, evaluation of telomerase activity, assessment of the gene expression profile and activity of major cancer pathways, cytogenetic analysis on synchronous MSCs, and molecular karyotyping using a comparative genomic hybridization (CGH) array.
Results
MSC-SAR displayed comparable morphology, immunophenotype, proliferation rate, differentiation potential, and telomerase activity to MSC-CTRL. Both cell types displayed signs of senescence in the late stages of culture with no relevant changes in cancer gene expression. However, cytogenetic analysis detected chromosomal anomalies in the early and late stages of MSC-SAR and MSC-CTRL after culture.
Conclusions
Our results demonstrated that the in vitro expansion of MSCs does not influence or favor malignant transformation since MSC-SAR were not more prone than MSC-CTRL to deleterious changes during culture. However, the presence of chromosomal aberrations supports rigorous phenotypic, functional and genetic evaluation of the biosafety of MSCs, which is important for clinical applications.
doi:10.1186/1479-5876-12-95
PMCID: PMC4022272  PMID: 24716831
Ewing sarcoma (EWS); Mesenchymal stem cells (MSCs); Osteosarcoma (OS); Tissue regeneration; Tumorigenic transformation
5.  Characterization and profiling of immunomodulatory genes of equine mesenchymal stromal cells from non-invasive sources 
Introduction
Mesenchymal stromal cells (MSCs) have been extensively studied for their promising capabilities in regenerative medicine. Although bone marrow is the best-known source for isolating equine MSCs, non-invasive alternative sources such as umbilical cord blood (UCB), umbilical cord matrix (UCM), and peripheral blood (PB) have also been reported.
Methods
Equine MSCs from three non-invasive alternative sources were isolated from six individual mares (PB) and their foals (UCB and UCM) at parturition. To minimize inter-horse variability, the samples from the three sources were matched within the same mare and for UCB and UCM even within the same foal from that specific mare. The following parameters were analyzed: (i) success rate of isolation, (ii) proliferation capacity, (iii) tri-lineage differentiation ability, (iv) immunophenotypical protein, and (v) immunomodulatory mRNA profiles. Linear regression models were fit to determine the association between the source of MSCs (UCB, UCM, PB) and (i) the moment of first observation, (ii) the moment of first passage, (iii) cell proliferation data, (iv) the expression of markers related to cell immunogenicity, and (v) the mRNA profile of immunomodulatory factors, except for hepatocyte growth factor (HGF) as no normal distribution could be obtained for the latter variable. To evaluate the association between the source of MSCs and the mRNA expression of HGF, the non-parametric Kruskal-Wallis test was performed instead.
Results
While equine MSCs could be isolated from all the UCB and PB samples, isolation from UCM was successful in only two samples because of contamination issues. Proliferation data showed that equine MSCs from all three sources could be easily expanded, although UCB-derived MSCs appeared significantly faster in culture than PB- or UCM-derived MSCs. Equine MSCs from both UCB and PB could be differentiated toward the osteo-, chondro-, and adipogenic lineage, in contrast to UCM-derived MSCs in which only chondro- and adipogenic differentiation could be confirmed. Regardless of the source, equine MSCs expressed the immunomodulatory genes CD40, CD80, HGF, and transforming growth factor-beta (TGFβ). In contrast, no mRNA expression was found for CD86, indoleamine 2,3-dioxygenase (IDO), and tumor necrosis factor-alpha (TNFα).
Conclusions
Whereas UCM seems less feasible because of the high contamination risks and low isolation success rates, UCB seems a promising alternative MSC source, especially when considering allogeneic MSC use.
doi:10.1186/scrt395
PMCID: PMC4055120  PMID: 24418262
6.  Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells 
Arthritis Research & Therapy  2012;14(3):R153.
Introduction
The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.
Methods
Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.
Results
Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.
Conclusions
Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.
doi:10.1186/ar3889
PMCID: PMC3446539  PMID: 22726892
7.  Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells 
Background
Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.
Results
Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.
Conclusion
Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.
doi:10.1186/1746-6148-8-150
PMCID: PMC3442961  PMID: 22937862
Canine; Mesenchymal stem cell; Cell surface markers; Embryonic stem cell markers
8.  Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia 
Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.
PMCID: PMC2945278  PMID: 20877435
Hypoxia; Wharton's jelly; Mesenchymal stem cells (MSCs); Transcription; Transformation markers; Cell proliferation.
9.  Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling 
Introduction
Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed as an ideal autologous stem cell source for cell-based therapy for myocardial infarction (MI). However, decreased viability and impaired function of aged MSCs hampered the therapeutic efficacy of engrafted MSCs, and the underlying mechanisms remain unclarified. Here, we investigated the role of inositol phosphates 6 kinase (IP6Ks) inhibition on the therapeutic efficacy of BM-MSCs and its underlying mechanism.
Methods
BM-MSCs isolated from young (8-week-old) or aged (18-month-old) donor male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury with or without administration of inositol phosphates 6 kinase (IP6Ks) inhibitor TNP (10 μM). MSC apoptosis induced by H/SD was determined by flow cytometry and TUNEL assays. Protein expressions were evaluated by Western blot assay. Furthermore, the paracrine effects of MSCs were measured by reverse transcriptase–polymerized chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses.
Results
Aged BM-MSCs exhibited more Inositol pyrophosphate 7 (IP7) production, compared with young BM-MSCs. Meanwhile, the expression of phospho-Akt (Thr308) was significantly decreased in the aged MSCs, resulting in enhanced Bad activation and decreased Bax/Bcl-2 ratio. Moreover, the apoptosis in aged BM-MSCs was increased, compared with young BM-MSCs. Furthermore, TNP administration significantly inhibited IP7 production and increased the phosphorylation of Akt under both normoxic and hypoxic conditions. Meanwhile, IP6Ks inhibition reduced apoptotic index of aged MSCs, associated with decreased expressions of pro-apoptotic proteins Bax and Bad and increased anti-apoptotic protein Bcl-2. The expressions of angiogenic factors, including VEGF, bFGF, IGF-1 and HGF, were decreased in MSCs from aged mice. In addition, TNP administration enhanced the paracrine efficiency of aged BM-MSCs under normoxic and hypoxic conditions.
Conclusions
This study demonstrates for the first time that IP6Ks and IP7 play critical role in the aging related vulnerability to hypoxic injury and impaired paracrine efficiency of BM-MSCs, which is associated with impaired Akt activation.
doi:10.1186/scrt431
PMCID: PMC4055148  PMID: 24670364
10.  Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts 
PLoS ONE  2011;6(9):e24164.
Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.
doi:10.1371/journal.pone.0024164
PMCID: PMC3170333  PMID: 21931658
11.  Lung-Derived Mesenchymal Stromal Cell Post-Transplantation Survival, Persistence, Paracrine Expression, and Repair of Elastase-Injured Lung 
Stem Cells and Development  2011;20(10):1779-1792.
While multipotent mesenchymal stromal cells have been recently isolated from adult lung (L-MSCs), there is very limited data on their biological properties and therapeutic potential in vivo. How L-MSCs compare with bone marrow-derived MSCs (BM-MSCs) is also unclear. In this study, we characterized L-MSC phenotype, clonogenicity, and differentiation potential, and compared L-MSCs to BM-MSCs in vivo survival, retention, paracrine gene expression, and repair or elastase injury after transplantation. L-MSCs were highly clonogenic, frequently expressed aldehyde dehydrogenase activity, and differentiated into osteocytes, chondrocytes, adipocytes, myofibroblasts, and smooth muscle cells. After intravenous injection (2 h), L-MSCs showed greater survival than BM-MSCs; similarly, L-MSCs were significantly more resistant than BM-MSCs to anchorage independent culture (4 h) in vitro. Long after transplantation (4 or 32 days), a significantly higher number of CD45neg L-MSCs were retained than BM-MSCs. By flow cytometry, L-MSCs expressed more intercellular adhesion molecule-1 (ICAM-1), platelet derived growth factor receptor alpha (PDGFRα), and integrin α2 than BM-MSCs; these proteins were found to modulate endothelial adherence, directional migration, and migration across Matrigel in L-MSCs. Further, L-MSCs with low ICAM-1 showed poorer lung retention and higher phagocytosis in vivo. Compared with BM-MSCs, L-MSCs expressed higher levels of several transcripts (e.g., Ccl2, Cxcl2, Cxcl10, IL-6, IL-11, Hgf, and Igf2) in vitro, although gene expression in vivo was increased by L-MSCs and BM-MSCs equivalently. Accordingly, both L-MSCs and BM-MSCs reduced elastase injury to the same extent. This study demonstrates that tissue-specific L-MSCs possess mechanisms that enhance their lung retention after intravenous transplantation, and produce substantial healing of elastase injury comparable to BM-MSCs.
doi:10.1089/scd.2011.0105
PMCID: PMC3182034  PMID: 21585237
12.  Adipose-derived mesenchymal stromal cells from genetically modified pigs: immunogenicity and immune modulatory properties 
Cytotherapy  2012;14(4):494-504.
Background aims
The immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSC) could prove to be a potential therapeutic approach for prolongation of survival of cell xenotransplantation. Adipose (Ad) MSC from genetically modified pigs could be an abundant source of pig donor-specific MSC.
Methods
Pig (p) MSC were isolated from adipose tissue of α1,3-galactosyltransferase gene knock-out pigs transgenic for human (h) CD46 (GTKO/ hCD46), a potential source of islets. After characterization with differentiation and flow cytometry (FCM), AdMSC were compared with bone marrow (BM) MSC of the same pig and human adipose-derived (hAd) MSC. The modulation of human peripheral blood mononuclear cell (hPBMC) responses to GTKO pig aortic endothelial cells (pAEC) by different MSC was compared by measuring 3H-thymidine uptake. The supernatants from the AdMSC cultures were used to determine the role of soluble factors.
Results
GTKO/hCD46 pAdMSC (i) did not express galactose-α1,3-galactose (Gal) but expressed hCD46, (ii) differentiated into chondroblasts, osteocytes and adipocytes, (iii) expressed stem cell markers, (iv) expressed lower levels of Swine Leucocyte Antigen I (SLAI), Swine Leucocyte Antigen II DR (SLAIIDR) and CD80 than pAEC before and after pig interferon (IFN)-γ stimulation. The proliferative responses of hPBMC to GTKO/hCD46 pAdMSC and hAdMSC stimulators were similar, and both were significantly lower than to GTKO pAEC (P < 0.05). The proliferation of hPBMC to GTKO pAEC was equally suppressed by GTKO/hCD46 pAdMSC and hAdMSC (P > 0.05). The supernatant from GTKO/hCD46 pAdMSC did not suppress the human xenoresponse to GTKO pAEC, which was cell–cell contact-dependent.
Conclusions
Initial evidence suggests that genetically modified pAdMSC function across the xenogeneic barrier and may have a role in cellular xenotransplantation.
doi:10.3109/14653249.2011.651529
PMCID: PMC3774176  PMID: 22264190
α1,3-galactosyl transferase knock-out; adipose-derived; mesenchymal stromal cells; pig; xenotransplantation
13.  Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses 
Background
Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses.
Results
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively.
Conclusions
Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.
doi:10.1186/1746-6148-9-133
PMCID: PMC3751243  PMID: 23826829
Mesenchymal stem cell; Bovine fetuses; Differentiation potential; Multipotency
14.  Prevention of LPS-Induced Acute Lung Injury in Mice by Mesenchymal Stem Cells Overexpressing Angiopoietin 1 
PLoS Medicine  2007;4(9):e269.
Background
The acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar–capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Current specific treatment strategies for ALI/ARDS are lacking. We hypothesized that mesenchymal stem cells (MSCs), with or without transfection with the vasculoprotective gene angiopoietin 1 (ANGPT1) would have beneficial effects in experimental ALI in mice.
Methods and Findings
Syngeneic MSCs with or without transfection with plasmid containing the human ANGPT1 gene (pANGPT1) were delivered through the right jugular vein of mice 30 min after intratracheal instillation of lipopolysaccharide (LPS) to induce lung injury. Administration of MSCs significantly reduced LPS-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts in bronchoalveolar lavage (BAL) fluid (53%, 95% confidence interval [CI] 7%–101%; and 60%, CI 4%–116%, respectively) as well as reducing levels of proinflammatory cytokines in both BAL fluid and lung parenchymal homogenates. Furthermore, administration of MSCs transfected with pANGPT1 resulted in nearly complete reversal of LPS-induced increases in lung permeability as assessed by reductions in IgM and albumin levels in BAL (96%, CI 6%–185%; and 74%, CI 23%–126%, respectively). Fluorescently tagged MSCs were detected in the lung tissues by confocal microscopy and flow cytometry in both naïve and LPS-injured animals up to 3 d.
Conclusions
Treatment with MSCs alone significantly reduced LPS-induced acute pulmonary inflammation in mice, while administration of pANGPT1-transfected MSCs resulted in a further improvement in both alveolar inflammation and permeability. These results suggest a potential role for cell-based ANGPT1 gene therapy to treat clinical ALI/ARDS.
Using a mouse model of acute respiratory distress syndrome, Duncan Stewart and colleagues report that rescue with mesenchymal stem cells expressing human angiopoietin 1 can avert lung injury from lipopolysaccharide.
Editors' Summary
Background.
Critically ill people who have had an injury to their lungs, for example through pneumonia, trauma, or an immune response to infection, may end up developing a serious complication in the lung termed acute respiratory distress syndrome (ARDS). In ARDS, inflammation develops in the lung, and fluid builds up in the alveoli (the air sacs resembling “bunches of grapes” at the ends of the network of tubes in the lung). This buildup of fluid prevents oxygen from being carried efficiently from air into the blood; the individual consequently experiences problems breathing and can develop further serious complications, which contribute significantly to the burden of illness among people in intensive care units. The death rate among individuals who do develop ARDS is very high, upward of 30%. Normally, individuals with ARDS are given extra oxygen, and may need a machine to help them breathe; treatments also focus on addressing the underlying causes in each particular patient. However, currently there are very few specific treatments that address ARDS itself.
Why Was This Study Done?
The researchers here wanted to work toward new treatment options for individuals with ARDS. One possible approach involves cells known as mesenchymal stem cells (MSCs). These cells are typically found in the bone marrow and have a property shared by very few other cell types in the body; they are able to carry on dividing and renewing themselves, and can eventually develop into many other types of cell. The researchers already knew that MSCs could become incorporated into injured lungs in mice and develop there into the tissue layers lining the lung. Some interesting work had also been done on a protein called angiopoeitin 1 (ANGPT1), which seemed to play a role in protecting against inflammation in blood vessels. Therefore, there was a strong rationale for carrying out experiments in mice to see if MSCs engineered to produce the ANGPT1 protein might “rescue” lung injury in mice. These experiments would be an initial step toward developing possible new treatments for humans with ARDS.
What Did the Researchers Do and Find?
The researchers used a mouse model to mimic the human ARDS condition. This involved injecting the windpipe of experimental mice with lipopolysaccharide (a substance normally found on the outer surface of bacteria that brings about an immune reaction in the lung). After 30 minutes, the mice were then injected with either salt solution (as a control), the MSCs, or MSCs producing the ANGPT1 protein. The researchers then looked at markers of lung inflammation, the appearance of the lungs under a microscope, and whether the injected MSCs had become incorporated into the lung tissue.
The lipopolysaccharide brought about a large increase in the number of inflammatory cells in the lung fluid, which was reduced in the mice given MSCs. Furthermore, in mice given the MSCs producing ANGPT1 protein, the number of inflammatory cells was reduced to a level similar to that of mice that had not been given lipopolysaccharide. When the researchers looked at the appearance under the microscope of lungs from mice that had been given lipopolysaccharide, they saw signs of inflammation and fluid coming out into the lung air spaces. These signs were reduced among both mice treated with MSCs and those treated with MSCs producing ANGPT1. The researchers also measured the “leakiness” of the lung tissues in lipopolysaccharide-treated mice; MSCs seemed to reduce the leakiness to some extent, and the lungs of mice treated with MSCs producing ANGPT1 were no more leaky than those of mice that had never been injected with lipopolysaccharide. Finally, the MSCs were seen to be incorporated into lung tissue by three days after injection, but after that were lost from the lung.
What Do These Findings Mean?
Previous research done by the same group had shown that fibroblasts producing ANGPT1 could prevent lung injury in rats later given lipopolysaccharide. The experiments reported here go a step further than this, and suggest that MSCs producing ANGPT1 can “rescue” the condition of mouse lungs that had already been given lipopolysaccharide. In addition, treatment with MSCs alone also produced beneficial effects. This opens up a possible new treatment strategy for ARDS in humans. However, it should be emphasized that the animal model used here is not a precise parallel of ARDS in humans, and that more research remains to be done before human studies of this sort could be considered.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040269.
Medline Plus entry on acute respiratory distress syndrome, providing basic information about what ARDS is, its effects, and how it is currently managed
ARDS Network from the US National Heart, Lung, and Blood Institute of the National Institutes of Health; the site provides frequently asked questions about ARDS as well as a list of clinical trials conducted by the network
Information about stem cells from the US National Institutes of Health, including information about the potential uses of stem cells
Wikipedia page about mesenchymal stem cells (note: Wikipedia is an internet encyclopedia anyone can edit)
doi:10.1371/journal.pmed.0040269
PMCID: PMC1961632  PMID: 17803352
15.  Bone marrow-derived mesenchymal stem cells promote growth and angiogenesis of breast and prostate tumors 
Introduction
Mesenchymal stem cells (MSCs) are known to migrate to tumor tissues. This behavior of MSCs has been exploited as a tumor-targeting strategy for cell-based cancer therapy. However, the effects of MSCs on tumor growth are controversial. This study was designed to determine the effect of MSCs on the growth of breast and prostate tumors.
Methods
Bone marrow-derived MSCs (BM-MSCs) were isolated and characterized. Effects of BM-MSCs on tumor cell proliferation were analyzed in a co-culture system with mouse breast cancer cell 4T1 or human prostate cancer cell DU145. Tumor cells were injected into nude mice subcutaneously either alone or coupled with BM-MSCs. The expression of cell proliferation and angiogenesis-related proteins in tumor tissues were immunofluorescence analyzed. The angiogenic effect of BM-MSCs was detected using a tube formation assay. The effects of the crosstalk between tumor cells and BM-MSCs on expression of angiogenesis related markers were examined by immunofluorescence and real-time PCR.
Results
Both co-culturing with mice BM-MSCs (mBM-MSCs) and treatment with mBM-MSC-conditioned medium enhanced the growth of 4T1 cells. Co-injection of 4T1 cells and mBM-MSCs into nude mice led to increased tumor size compared with injection of 4T1 cells alone. Similar experiments using DU145 cells and human BM-MSCs (hBM-MSCs) instead of 4T1 cells and mBM-MSCs obtained consistent results. Compared with tumors induced by injection of tumor cells alone, the blood vessel area was greater in tumors from co-injection of tumor cells with BM-MSCs, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. Furthermore, both conditioned medium from hBM-MSCs alone and co-cultures of hBM-MSCs with DU145 cells were able to promote tube formation ability of human umbilical vein endothelial cells. When hBM-MSCs are exposed to the DU145 cell environment, the expression of markers associated with neovascularization (macrophage inflammatory protein-2, vascular endothelial growth factor, transforming growth factor-beta and IL-6) was increased.
Conclusion
These results indicate that BM-MSCs promote tumor growth and suggest that the crosstalk between tumor cells and BM-MSCs increased the expression of pro-angiogenic factors, which may have induced tumor cell proliferation and angiogenesis thereby increasing solid tumor growth.
doi:10.1186/scrt221
PMCID: PMC3707041  PMID: 23763837
Mesenchymal Stem Cells; Tumor Growth; Angiogenesis
16.  Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation, composition, and micromechanical properties of nucleus pulposus constructs 
Introduction
Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs.
Methods
Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-β3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM).
Results
GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-β or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs.
Conclusions
The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.
doi:10.1186/ar4505
PMCID: PMC4060243  PMID: 24618041
17.  Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status 
BMC Cell Biology  2011;12:12.
Background
In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.
Results
Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity".
Conclusion
The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.
doi:10.1186/1471-2121-12-12
PMCID: PMC3073900  PMID: 21450070
18.  Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats 
Background
Several lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.
Results
Isolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.
Conclusions
Our data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.
doi:10.1186/1423-0127-21-5
PMCID: PMC3922747  PMID: 24447306
Mesenchymal stem cells; Conditioned medium; Neuronal cultures; Ischemic stroke; Neuroprotection; Cell surface markers
19.  Comprehensive Characterization of Mesenchymal Stem Cells from Human Placenta and Fetal Membrane and Their Response to Osteoactivin Stimulation 
Stem Cells International  2012;2012:658356.
Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC) and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC) and fetal membrane (Mb-MSC) through morphological and fluorescent-activated cell sorting (FACS). MSC frequency is higher in membrane than placenta (2.14%  ± 0.65 versus 15.67%  ± 0.29%). Pl/Mb-MSCs in vitro expansion potential was significantly higher than BM-MSCs. We demonstrated that one of the MSC-specific marker is sufficient for MSC isolation and that culture in specific media is the optimal way for selecting very homogenous MSC population. These MSCs could be differentiated into mesodermal cells expressing cell markers and cytologic staining consistent with mature osteoblasts and adipocytes. Transcriptomic analysis and cytokine arrays demonstrated broad similarity between placenta- and membrane-derived MSCs and only discrete differences with BM-MSCs with enrichment of networks involved in bone differentiation. Pl/Mb-MSCs displayed higher osteogenic differentiation potential than BM-MSC when their response to osteoactivin was evaluated. Fetal-tissue-derived mesenchymal cells may, therefore, be considered as a major source of MSCs to reach clinical scale banking in particular for bone regeneration.
doi:10.1155/2012/658356
PMCID: PMC3373208  PMID: 22701494
20.  Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses 
BMC Veterinary Research  2014;10:154.
Background
Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses.
Results
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes.
Conclusion
The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.
doi:10.1186/1746-6148-10-154
PMCID: PMC4098697  PMID: 25011474
Bovine fetuses; Mesenchymal stem cell; Differentiation potential; Hepatocyte-like cell; Neuron-like cell
21.  Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells 
BMC Medicine  2013;11:146.
Background
Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties.
Methods
BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells.
Results
BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs.
Conclusions
By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.
doi:10.1186/1741-7015-11-146
PMCID: PMC3694028  PMID: 23758701
Mesenchymal stromal/stem cells; Age; Gender; Immunomodulation; Phenotype; Differentiation
22.  Characterization of Human Mesenchymal Stem Cells from Ewing Sarcoma Patients. Pathogenetic Implications 
PLoS ONE  2014;9(2):e85814.
Background
Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin.
Materials and Methods
In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples.
Results
We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99.
Conclusions
In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis.
doi:10.1371/journal.pone.0085814
PMCID: PMC3911896  PMID: 24498265
23.  Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin 
Stem Cells and Development  2011;21(10):1761-1768.
Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription–polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow–derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue–derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.
doi:10.1089/scd.2011.0030
PMCID: PMC3376459  PMID: 21970410
24.  Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells 
Introduction
The ability to self-renew, be easily expanded in vitro and differentiate into different mesenchymal tissues, render mesenchymal stem cells (MSCs) an attractive therapeutic method for degenerative diseases. The subsequent discovery of their immunosuppressive ability encouraged clinical trials in graft-versus-host disease and auto-immune diseases. Despite sharing several immunophenotypic characteristics and functional capabilities, the differences between MSCs arising from different tissues are still unclear and the published data are conflicting.
Methods
Here, we evaluate the influence of human MSCs derived from umbilical cord matrix (UCM), bone marrow (BM) and adipose tissue (AT), co-cultured with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNC), on T, B and natural killer (NK) cell activation; T and B cells’ ability to acquire lymphoblast characteristics; mRNA expression of interleukin-2 (IL-2), forkhead box P3 (FoxP3), T-bet and GATA binding protein 3 (GATA3), on purified T cells, and tumor necrosis factor-alpha (TNF-α), perforin and granzyme B on purified NK cells.
Results
MSCs derived from all three tissues were able to prevent CD4+ and CD8+ T cell activation and acquisition of lymphoblast characteristics and CD56dim NK cell activation, wherein AT-MSCs showed a stronger inhibitory effect. Moreover, AT-MSCs blocked the T cell activation process in an earlier phase than BM- or UCM-MSCs, yielding a greater proportion of T cells in the non-activated state. Concerning B cells and CD56bright NK cells, UCM-MSCs did not influence either their activation kinetics or PHA-induced lymphoblast characteristics, conversely to BM- and AT-MSCs which displayed an inhibitory effect. Besides, when co-cultured with PHA-stimulated MNC, MSCs seem to promote Treg and Th1 polarization, estimated by the increased expression of FoxP3 and T-bet mRNA within purified activated T cells, and to reduce TNF-α and perforin production by activated NK cells.
Conclusions
Overall, UCM-, BM- and AT-derived MSCs hamper T cell, B cell and NK cell-mediated immune response by preventing their acquisition of lymphoblast characteristics, activation and changing the expression profile of proteins with an important role in immune function, except UCM-MSCs showed no inhibitory effect on B cells under these experimental conditions. Despite the similarities between the three types of MSCs evaluated, we detect important differences that should be taken into account when choosing the MSC source for research or therapeutic purposes.
doi:10.1186/scrt336
PMCID: PMC3854702  PMID: 24406104
25.  Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells1 
Cell medicine  2012;4(1):1-11.
Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and secrete mediators in response to activation. Five MSC lines from each tissue were isolated. Lymphocyte proliferation was assessed in a mixed leukocyte reaction, and mediator secretion was determined by ELISA. Regardless of tissue of origin, quiescent MSCs did not alter lymphocyte proliferation or secrete mediators, except for transforming growth factor-β (TGF-β1). When stimulated, MSCs of all tissue types decreased lymphocyte proliferation, increased prostaglandin (PGE2) and interleukin-6 (IL-6) secretion, and decreased production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BM-MSCs and CB-MSCs also produced nitric oxide (NO), while AT-MSCs and CT-MSCs did not. Equine MSCs did not produce indoleamine 2,3-dioxygenase (IDO). These data suggest that activated equine MSCs derived from BM, AT, CT, and CB secrete high concentration of mediators and are similar to MSCs from rodents and humans in their immunomodulatory profiles. These findings have implication for the treatment of inflammatory lesions dominated by activated lymphocytes and TNF-α and IFN-γ in vivo.
doi:10.3727/215517912X647217
PMCID: PMC3495591  PMID: 23152950
Equine; Mesenchymal stem cells; Immunomodulation; Lymphocytes; Bone marrow; Umbilical cord blood; Adipose and umbilical cord tissue

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