Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy in regenerative medicine. To date, MSCs have been obtained from conventional bone marrow via a highly invasive procedure. Therefore, MSCs are now also isolated from sources such as adipose tissue, cord blood and cord stroma, a subject of growing interest. As the characterization and differentiation potential of adipose-derived MSCs (AD-MSCs) and bone-marrow-derived MSCs (BM-MSCs) have not been documented, we have evaluated and compared the characteristics of both MSC types by qualitative and quantitative analyses. Both cell types show similar morphology and surface protein expression, being positive for stromal-associated markers and negative for hematopoietic and endothelial markers. The colony-forming potential of AD-MSCs is distinctly higher than that of BM-MSCs. Nonetheless, similar adipogenic and osteogenic differentiation is observed in both groups of MSCs. Cytochemical qualitative analysis and calcium mineralization demonstrate higher levels toward osteogenic differentiation in BM-MSCs than in AD-MSCs. On the contrary, the percentage of Nile red oil staining for differentiated adipocytes is higher in AD-MSCs than in BM-MSCs. Quantitative real-time polymerase chain reaction shows similar patterns of osteogenic- and adipogenic-associated gene expression in both cell types. Each of the MSCs respond in functional analysis by exhibiting unique properties at the differentiation level according to their micro-environmental niche. Thus, quantitative analysis might be a valuable means of describing stem cell multipotency, in addition to qualitative investigation.     

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid, β-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro.In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type 1 appeared to be influenced by the presence of dexamethasone.     

Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow

Embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) have been studied for years as primary cell sources for regenerative biology and medicine. MSCs have been derived from cell and tissue sources, such as bone marrow (BM), and more recently from ESCs. This study investigated MSCs derived from BM, H1- and H9-ESC lines in terms of morphology, surface marker and growth factor receptor expression, proliferative capability, modulation of immune cell growth and multipotency, in order to evaluate ESC-MSCs as a cell source for potential regenerative applications. The results showed that ESC-MSCs exhibited spindle-shaped morphology similar to BM-MSCs but of various sizes, and flow cytometric immunophenotyping revealed expression of characteristic MSC surface markers on all tested cell lines except H9-derived MSCs. Differences in growth factor receptor expression were also shown between cell lines. In addition, ESC-MSCs showed greater capabilities for cell proliferation, and suppression of leukocyte growth compared to BM-MSCs. Using standard protocols, induction of ESC-MSC differentiation along the adipogenic, osteogenic, or chondrogenic lineages was less effective compared to that of BM-MSCs. By adding bone morphogenetic protein 7 (BMP7) into transforming growth factor beta 1 (TGFβ1)-supplemented induction medium, chondrogenesis of ESC-MSCs was significantly enhanced. Our findings suggest that ESC-MSCs and BM-MSCs show differences in their surface marker profiles and the capacities of proliferation, immunomodulation, and most importantly multi-lineage differentiation. Using modified chondrogenic medium with BMP7 and TGFβ1, H1-MSCs can be effectively induced as BM-MSCs for chondrogenesis.     

Platelet lysate suppresses the expression of lipocalin-type prostaglandin D2 synthase that positively controls adipogenic differentiation of human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have been shown to display a considerable therapeutic potential in cellular therapies. However, harmful adipogenic maldifferentiation of transplanted MSCs may seriously threaten the success of this therapeutic approach. We have previously demonstrated that using platelet lysate (PL) instead of widely used fetal calf serum (FCS) diminished lipid accumulation in adipogenically stimulated human MSCs and identified, among others, lipocalin-type prostaglandin D2 synthase (L-PGDS) as a gene suppressed in PL-supplemented MSCs. Here, we investigated the role of PL and putatively pro-adipogenic L-PGDS in human MSC adipogenesis. Next to strongly reduced levels of L-PGDS we show that PL-supplemented MSCs display markedly decreased expression of adipogenic master regulators and differentiation markers, both before and after induction of adipocyte differentiation. The low adipogenic differentiation capability of PL-supplemented MSCs could be partially restored by exogenous addition of L-PGDS protein. Conversely, siRNA-mediated downregulation of L-PGDS in FCS-supplemented MSCs profoundly reduced adipocyte differentiation. In contrast, inhibiting endogenous prostaglandin synthesis by aspirin did not reduce differentiation, suggesting that a mechanism such as lipid shuttling but not the prostaglandin D2 synthase activity of L-PGDS is critical for adipogenesis. Our data demonstrate that L-PGDS is a novel pro-adipogenic factor in human MSCs which might be of relevance in adipocyte metabolism and disease. L-PGDS gene expression is a potential quality marker for human MSCs, as it might predict unwanted adipogenic differentiation after MSC transplantation.     

Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen.     

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.     

Isolation and characterization of mesenchymal stem cell population entrapped in bone marrow collection sets

Bone marrow is an important source of mesenchymal stem cells (MSCs), and a promising tool for cytotherapy. MSC utilization is limited by low cell yields obtained under standard isolation protocols. Herein, used bone marrow collection sets were evaluated as a valuable source of MSCs. Adherent cells washed from the collection sets were examined for widely accepted criteria defining MSCs. Significant numbers of cells (median 9million per set in passage 1) with colony-forming activity and high proliferative potential at low seeding densities were obtained. These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29) and negative for most haematopoietic and endothelial cell markers (CD45, CD34, CD11a, CD235a, HLA-DR, CD144). The cells were capable of differentiation along adipogenic, osteogenic and chondrogenic pathways. Washing out bone marrow collection sets may constitute a highly ethical source of MSCs for research purposes and may be utilized also in clinical applications.     

Adipocyte lineage differentiation potential of MSCs isolated from reaming material

Mesenchymal stem cells (MSCs) obtained from various sources have been used for different therapeutic applications including tissue regeneration. Reamer/irrigator/aspirator (RIA) has been increasingly used in recent years for the derivation of MSCs. Here in this investigation we have comparatively analyzed MSCs obtained from iliac crest bone marrow (ICBM) and RIA for their morphology, cluster determinant (CD) markers, and adipogenic differentiation capacity. MSCs were isolated, cultured, and purified from both sources and then flow cytometric studies were performed to study their characteristics. The differentiation potential of RIA and ICBM was examined by an Oil Red O staining protocol. Moreover, the tissue-specific markers related to adipogenesis were analyzed by real-time polymerase chain reaction (RT-PCR). The cells were cultured in the relevant induction medium and then adipogenic lineage differentiation was tested and confirmed for all MSC preparations. Additionally, analysis by flow cytometer was indicative of RIA derived MSCs (RIA-MSCs) having a more homogenous population than ICBM derived MSCs. The RIA-MSCs differentiation toward adipogenic lineage was more efficient compared with ICBM-MSCs. Direct comparative analysis of RIA to ICBM-MSCs indicated that the RIA-MSCs had a higher potential toward adipocyte lineage differentiation compared with ICBM-MSCs.     

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.     

Osteogenic growth peptide C-terminal pentapeptide [OGP(10-14)] acts on rat bone marrow mesenchymal stem cells to promote differentiation to osteoblasts and to inhibit differentiation to adipocytes

Cumulative evidence indicates that bone marrow mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating to osteogenic and adipogenic lineages when stimulated under appropriate conditions. Whether OGP(10-14) directly regulates the progenitor cells differentiating into osteoblasts or adipocytes remains unknown. In the present study, we investigated the roles of OGP(10-14) in differentiation along these separate lineages using rat bone marrow MSCs. Our results showed that OGP(10-14) promoted osteogenic differentiation of the stem cells and concurrently inhibited adipocyte formation. OGP(10-14) increased alkaline phosphatase (ALP) activity and mineralized nodule formation, and stimulated osteoblast-specific mRNA expression of core-binding factor 1 (cbfa1). In contrast, OGP(10-14) decreased adipocyte numbers and inhibited adipocyte-specific mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2). These observations suggest that commitment of MSCs into osteogenic or adipogenic lineages is regulated by OGP(10-14).     

High affinity leptin receptors are present in human mesenchymal stem cells (MSCs) derived from control and osteoporotic donors

There are disparate observations on central and peripheral effects of leptin, but several studies consistently support its role as a link between fat and bone. Bone marrow stroma contains mesenchymal stem cells (MSCs), which differentiate into osteoblasts and adipocytes, among others. In this study we assessed the expression of leptin receptors protein in MSCs from control and osteoporotic postmenopausal donors and their change during osteogenic and adipogenic differentiation. Also, we assessed the effects of leptin on osteogenic and adipogenic differentiation of these cells. We demonstrated high affinity leptin binding (KD = 0.36 +/- 0.02 nM) in both types of cells. Binding was very low under basal, but increased significantly (2-3 times) through osteogenic and adipogenic differentiation. Osteoporotic MSCs showed lower leptin binding capacity than control cells at an early osteogenic and adipogenic differentiation time, which could restrict cell sensitivity to the protective action of leptin. In this regard, we observed that leptin significantly inhibited adipocyte differentiation in control but not in osteoporotic MSCs, while it exerted a low stimulatory effect on calcium deposition (10%-20%) in both types of MSCs cells. In summary, we report the presence of high affinity leptin receptors on control and osteoporotic MSCs, which were modified distinctly by osteogenic and adipogenic stimulation and a direct and distinct effect of leptin on both type of cells.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.