Mesenchymal Stem Cells Specific Markers Research Articles

Isolation and Characterization of Mesenchymal Stem Cells from Canine Ovarian Surface Epithelium

Background: Few studies have confirmed the presence of ovarian tissue stem cells indicating the capacity for differentiation. Based on this fact, it was hypothesized that mesenchymal stem cells (MSC) were found in ovarian surface epithelium (OSE) of canines that could easily be isolated. Methods: Both left and right ovaries were minced and digested using collagenase to obtain a stromal vascular fraction (SVF). MSCs were characterized using RT-PCR. To ascertain the trilineage differentiation potential, MSCs were stained with respective stain for osteocytes, chondrocytes and adipocytes. Result: We observed elongated, spindle-shaped and fibroblast like appearance of cells after 72 h of initial culture. Expression of MSC specific surface markers were observed through RT-PCR. Using Stem Pro® differentiation medium, OSE were differentiated into osteogenic, chondrogenic and adipogenic lineages and were found to be potential source for isolation, characterization and differentiation of MSCs. Canine (OSE) is easily accessible, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

Optogenetic control of neural differentiation in Opto-mGluR6 engineered retinal pigment epithelial cell line and mesenchymal stem cells.

In retinal degenerative disorders, when neural retinal cells are damaged, cell transplantation is one of the most promising therapeutic approaches. Optogenetic technology plays an essential role in the neural differentiation of stem cells via membrane depolarization. This study explored the efficacy of blue light stimulation in neuroretinal differentiation of Opto-mGluR6-engineered mouse retinal pigment epithelium (mRPE) and bone marrow mesenchymal stem cells (BMSCs). mRPE and BMSCs were selected for optogenetic study due to their capability to differentiate into retinal-specific neurons. BMSCs were isolated and phenotypically characterized by the expression of mesenchymal stem cell-specific markers,CD44 (99%) andCD105 (98.8%). mRPE culture identity was confirmed by expression of RPE-specific marker, RPE65, and epithelial cell marker, ZO-1. mRPE cells and BMSCs were transduced with AAV-MCS-IRES-EGFP-Opto-mGluR6 viral vector and stimulated for 5 days with blue light (470 nm). RNA and protein expression of Opto-mGluR6 were verified. Optogenetic stimulation-induced elevated intracellular Ca2+ levels in mRPE- and BMS-treated cells. Significant increase in cell growth rate and G1/S phase transition were detected in mRPE- and BMSCs-treated cultures. Pou4f1, Dlx2, Eomes, Barlh2, Neurod2, Neurod6, Rorb, Rxrg, Nr2f2, Ascl1, Hes5, and Sox8 were overexpressed in treated BMSCs and Barlh2, Rorb, and Sox8 were overexpressed in treated mRPE cells. Expression of Rho, Thy1, OPN1MW, Recoverin, and CRABP, as retinal-specific neuron markers, in mRPE and BMS cell cultures were demonstrated. Differentiation of ganglion, amacrine, photoreceptor cells, and bipolar and Muller precursors were determined in BMSCs-treated culture and were compared with mRPE. mRPE cells represented more abundant terminal Muller glial differentiation compared with BMSCs. Our results also demonstrated that optical stimulation increased the intracellular Ca2+ level and proliferation and differentiation of Opto-mGluR6-engineered BMSCs. It seems that optogenetic stimulation of mRPE- and BMSCs-engineered cells would be a potential therapeutic approach for retinal degenerative disorders.

Subacromial Bursa–Derived Cells Demonstrate High Proliferation Potential Regardless of Patient Demographics and Rotator Cuff Tear Characteristics

To investigate the influence of patient demographics and rotator cuff tear characteristics on the cellular proliferation potential of subacromial bursa-derived cells (SBDCs). Patients undergoing arthroscopic rotator cuff repair between December 2017 and February 2019 were considered for enrollment in the study. Basic demographic information as well as medical and surgical history were obtained for each patient. Subacromial bursa was harvested from over the rotator cuff tendon. Cellular proliferation was evaluated after 3 weeks of incubation by counting nucleated SBDCs. Fluorescence-activated cell sorting (FACS) analysis was performed to confirm the presence of mesenchymal stem cell (MSC) specific surface markers. Using preoperative radiographs and magnetic resonance imaging (MRI), acromiohumeral distance (AHD), severity of cuff tear arthropathy, and rotator cuff tear characteristics were evaluated. Seventy-three patients (mean age: 57.2 ± 8.5 years) were included in the study. There was no significant difference in cellular proliferation of SBDCs when evaluating the influence of age, sex, body mass index (BMI), smoking status, and presence of systemic comorbidities (p > .05, respectively). Similarly, there was no significant difference in cellular proliferation of SBDCs when looking at rotator cuff tear characteristics (size, tendon retraction, fatty infiltration, muscle atrophy), AHD, or severity of cuff tear arthropathy (p > .05). FACS analysis confirmed nucleated SBDCs to have a high positive rate of MSC specific surface markers. Subacromial bursa consistently demonstrated a high cellular proliferation potential regardless of patient demographics, rotator cuff tear characteristics, and severity of glenohumeral joint degeneration. These findings may alleviate concerns that subacromial bursa might lose cellular proliferation potential when being used for biologic augmentation in massive and degenerated rotator cuff tears, thus assisting in predicting tendon healing and facilitating surgical decision-making.

Osteogenic potential of mesenchymal stem cells from rat mandible to regenerate critical sized calvarial defect

Although bone marrow–derived mesenchymal stem cells (MSCs) have been extensively explored in bone tissue engineering, only few studies using mesenchymal stem cells from mandible (M-MSCs) have been reported. However, mesenchymal stem cells from mandible have the potential to be as effective as femur-derived mesenchymal stem cells (F-MSCs) in regenerating bone, especially in the orofacial regions, which share embryonic origin, proximity, and accessibility. M-MSCs were isolated and characterized using mesenchymal stem cell–specific markers, colony forming assay, and multi-potential differentiation. In vitro osteogenic potential, including proliferation, osteogenic gene expression, alkaline phosphatase activity, and mineralization, was examined and compared. Furthermore, in vivo bone formations of F-MSCs and M-MSCs in rat critical sized defect were evaluated using microCT and histology. M-MSCs from rat could be successfully isolated and expanded while preserving their MSC’s characteristics. M-MSCs demonstrated a comparable proliferation and mineralization potentials and in vivo bone formation as F-MSCs. M-MSCs is a promising cell source candidate for craniofacial bone tissue engineering.

RNA-Sequencing Gene Expression Profiling of Orbital Adipose-Derived Stem Cell Population Implicate HOX Genes and WNT Signaling Dysregulation in the Pathogenesis of Thyroid-Associated Orbitopathy.

PurposeThe purpose of this study was to characterize the intrinsic cellular properties of orbital adipose-derived stem cells (OASC) from patients with thyroid-associated orbitopathy (TAO) and healthy controls.MethodsOrbital adipose tissue was collected from a total of nine patients: four controls and five patients with TAO. Isolated OASC were characterized with mesenchymal stem cell–specific markers. Orbital adipose-derived stem cells were differentiated into three lineages: chondrocytes, osteocytes, and adipocytes. Reverse transcription PCR of genes involved in the adipogenesis, chondrogenesis, and osteogenesis pathways were selected to assay the differentiation capacities. RNA sequencing analysis (RNA-seq) was performed and results were compared to assess for differences in gene expression between TAO and controls. Selected top-ranked results were confirmed by RT-PCR.ResultsOrbital adipose-derived stem cells isolated from orbital fat expressed high levels of mesenchymal stem cell markers, but low levels of the pluripotent stem cell markers. Orbital adipose-derived stem cells isolated from TAO patients exhibited an increase in adipogenesis, and a decrease in chondrogenesis and osteogenesis. RNA-seq disclosed 54 differentially expressed genes. In TAO OASC, expression of early neural crest progenitor marker (WNT signaling, ZIC genes and MSX2) was lost. Meanwhile, ectopic expression of HOXB2 and HOXB3 was found in the OASC from TAO.ConclusionOur results suggest that there are intrinsic genetic and cellular differences in the OASC populations derived from TAO patients. The upregulation in adipogenesis in OASC of TAO may be is consistent with the clinical phenotype. Downregulation of early neural crest markers and ectopic expression of HOXB2 and HOXB3 in TAO OASC demonstrate dysregulation of developmental and tissue patterning pathways.

ID: 1032 Differentiation Potential and Characteristics of Mesenchymal Stem Cells Isolated from Human Umbilical Cord membrane to Hepatocyte-like Cells

Recent studies indicated that Mesenchymal stem cell has become a potential objective for therapy. In this study, umbilical cord cells were isolated and analyzed the expression of mesenchymal stem cells specific markers then they were differentiated into hepatocyte-like cells by DMSO and Gene transfection. Umbilical cord mesenchymal stem cell (MSC) was isolated by explant culture in media DMEM/F12, complementing with growth factors EGF, FGF and IST. After that, they were exposured to DMSO with three concentrations: 0.01%, 0.1%, 1% and another group was transfection with HNF4α by Lipofectamin LX plus. The cells were analyzed at 1, 2, 3, and 4 weeks after treatment. The cells isolation was shown the positive with markers CD73, CD34, CD86, CD90, CD105, eras, Oct 1, GATA, and negative with markers HNF4α, Alb and G6P. In group 0,1% DMSO treatment, after 3 weeks the cells were positive with markers HNF4α but it was also negative with markers Alb and G6P. In the transfection group, the cell expresses HNF4α at three weeks after treatment. Although our results exposure that the umbilical cord mesenchymal stem cells expressed hepatic specific marker after DMSO induced and DNA treatment. So it will be necessary to optimize research conditional and investigate the hepatic functions of these cells in a longer in vitro culture.

Isolation, differentiation, and characterization of mesenchymal stem cells from human bone marrow.

We describe the minimum requirements and a simplified method for isolation and characterization of mesenchymal stem cells (MSCs) from human bone marrow. MSCs are well known adult stem cells present in many tissues such as adipocytes, chondrocytes, osteoblasts, and neurons. Many isolations and characterization methods have emerged to apply MSCs in the clinical applications, which many of them are expensive and time-consuming. MSC isolation was carried out from human bone marrow, and cultured in defined medium. Cultures were maintained at 370C in a humidified atmosphere containing 5% CO2 for 48h. The medium was exchanged every 3-4 days. Adherent cells were characterized according to main criteria defined by ISCT, such as differentiation capability to adipocyte and osteoblast using specific differentiation mediums; also, flow cytometry verified MSC specific markers. Isolated MSCs had a fibroblastic-like appearance with adherent property to the culture plate. Differentiation function was proved with the formation of lipid drops and calcium oxalates on the differentiated MSCs and finally, purified MSCs from bone marrow were positive for cell surface markers, CD73, CD90, and CD105 while being negative for CD34 and CD45. These findings confirm that the represented method is capable of isolating MSCs from bone marrow with proven results according to all minimum criteria defined by the International Society for Cellular Therapy (ISCT).

Tumor Specific Recruitment and Reprogramming of Mesenchymal Stem Cells in Tumorigenesis.

Non-neoplastic stromal cells harvested from patient tumors were identified as tumor-derived mesenchymal stem cells (MSCs) by their multipotential capacity to differentiate into adipocytes, osteoblasts, and chondrocytes and by the expression of MSC specific cell surface markers. These procedures yielded also epithelial cancer cells and their counterpart MSC from gastric carcinoma (GSC1) and lung carcinoma (LC2). While the LC2 cancer cell growth is independent of their LC-MSC, the GSC1 cancer cell growth is critically dependent on the presence of their counterpart GSC-MSC or their conditioned medium (CM). The fact that none of the various other tumor-derived MSCs was able to restore the specific effect of GSC-MSC on GSC1 cancer cell growth suggests specificity of tumor-derived MSC, which are specifically recruited and "educated"/reprogrammed by the cancer cells to support tumor growth. Using cytokine array analysis, we were able to demonstrate that GSC1 cell growth is mediated through hepatocyte growth factor (HGF)/c-MET signaling pathway which is activated exclusively by HGF secreted from GSC-MSC. An innovative approach demonstrates GSC1-mediated specific tropism of "naïve" MSC from the adjacent tissue in a tumor specific manner to support tumor progression. The results suggest that specific tumor tropic "naïve" MSC are reprogrammed in a tumor-specific manner to support gastric tumor progression. Understanding the mechanisms involved in the interactions of the tumor cancer cells and tumor-derived MSC will constitute the basis for developing multimodal anticancer therapeutic strategies that will also take into account the specific tumor tropism properties of MSC and their reprogramming.

The effect dmso on differentiation into hepatocytes-like cell of umbilical cord stem cells

Recent studies indicated that DMSO can induce differentiation of mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord blood, umbilical cord tissues into hepatocyte-like cells. However, the effect of DMSO is not similar in reports. In this study, umbilical cord cells were isolated and analyzed the expression of mesenchymal stem cells specific markers then they were exposured to DMSO with three concentrations: 0.01%, 0.1%, 1% within 3 days. Expression of specific markers was analyzed by RT-PCR after 10 days, 20 days, 30 days to investigate the differentiation level. The result showed that the isolated cells were positive with mesenchymal stem cells specific markers, such as: CD73, CD90, CD105. This expression is stable after 20 times of subculture. At the concentration 1% of DMSO, in the day 20-30th of in vitro culture, isolated cells expressed HNF4α gene, a transcription regulation factor in hepatocyte production. Conclusion: In the early part of this study, the umbilical cord mesenchymal stem cells expressed hepatic specific marker after DMSO treatment. It is necessary to investigate the hepatic functions of these cells in a longer in vitro culture.

Generation of handmade cloned embryos from adipose tissue derived mesenchymal stem cells in goat

The present study aims to isolate and culture adult mesenchymal stem cells (MSCs) from adult adipose tissues of goat followed by their thorough characterization and in vitro differentiation into osteocytes, chondrocytes and adipocytes. These cells were further used as donor nuclei for production of handmade cloned embryos. The goat adipose tissue-derived mesenchymal stem cells (ADSCs) were positive for the expression of CD29, CD34, CD44, CD90 and CD166 (MSCs specific positive markers) and negative for CD45 and CD71 (MSCs specific negative markers). The expression of specific markers for osteocytes (Osteopontin, Collagen I), chondrocytes (Aggrecan and Collagen II) and adipocytes (LPL, PPARα and PPARγ) was also detected from these cells. The present study reported no significant difference in cleavage, morulae and blastocyst production, when ADSCs (69.48±0.86, 30.69±1.09 and 10.96±0.82), fetal (73.3±1.26, 34.12±0.79 and 13.86±0.69) and adult (71.81±0.64, 34.07±1.00 and 11.71±0.63) fibroblasts were used as somatic donor cells. On staining with Hoechst 33342 dye, a well-developed blastocyst revealed 90–100 numbers of blastomeres under an epifluorescence microscope. In the future, the ADSCs may be used as a convenient source for autologous therapy and production of transgenic animals.

Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation.

Amnion-derived mesenchymal stem cells (AMSCs) are multipotent cells with an enhanced ability to differentiate into multiple lineages. AMSCs can be acquired through noninvasive methods, and therefore are exempt from the typical ethical issues surrounding stem cell use. The objective of this study was to isolate and characterize AMSCs from a cat amniotic membrane for future application in regenerative medicine. The cat AMSCs were harvested after mechanical and enzymatic digestion of amnion. In culture medium, the cat AMSCs adhered to a plastic culture dish and displayed a fibroblast-like morphology. Immunophenotyping assays were positive for the mesenchymal stem cell-specific markers CD73 and CD90 but not the hematopoietic markers CD34, CD45, and CD79. Under appropriate conditions, the cat AMSCs differentiated into osteogenic, chondrogenic, and adipogenic cell lineages. One advantage of cat AMSCs was nonteratogenicity, assessed 4 weeks post injection of undifferentiated AMSCs into immunodeficient mice. These findings suggest that cat amniotic membranes may be an important and useful source of mesenchymal stem cells for clinical applications, especially for cell or tissue replacement in chronic and degenerative diseases.

중간엽줄기세포 증식을 위한 누에 실샘 유래 무혈청 배지 첨가제

Mesenchymal stem cells (MSCs) are must be cultured in chemically defined, xeno-free culture system for cell-based therapies. One major overcome for their clinical use is the unsafety of fetal bovine serum (FBS), which is a crucial part of all media currently used for the culture of MSCs. Previously, we developed novel methods the production of silkworm gland hydrolysate (SGH). Also, we showed the effect of the SGH on the cell growth promotion. We demonstrated whether the characteristics of MSCs were maintained in the serum-free media containing SGH during long-term cultivation. The results of microscopic observation showed that the morphology of stem cells were not different between the media containing 10% FBS and 1 mg/mL SGH. After the long-term culture of MSCs in serum-free media containing SGH, the rate of MSCs growth was well maintained. The analysis of anti-apoptotic effect of SGH showed that early apoptosis of MSCs was reduced by 3.7-fold. Furthermore, MSCs specific surface markers and stemness gene expression (Nanog, Sox2) were preserved as that of MSCs in the media containing 10% FBS. In conclusion, we demonstrate that the SGH can be used as useful supplement for MSCs cultivation in serumfree media.

204 ISOLATION, CHARACTERIZATION, AND DIFFERENTIATION OF ADIPOSE TISSUE DERIVED MESENCHYMAL STEM CELLS: AN AUTOLOGOUS TRANSPLANTATION TO PATIENTS

Adult stem cells derived from all possible sources of livestock serve as the best possible alternative to embryonic stem cells. The discovery of mesenchymal stem cells has provided the new horizon to stem cell therapy. Adipose tissue derived mesenchymal stem cell (ADSCs), an easy source of adult stem cell has created a lot of interest among researchers as patient specific treatment and autologous transplantation in animals is becoming a viable option. The proposed study was carried out for 1) isolation of ADSCs from dogs, suffering from hip dysplasia or from paraplegia, 2) ADSC characterisation and in vitro differentiation ability into osteocytes, chondrocytes, adipocytes and neurocytes specific cells. Adipose tissues were collected from belly/umbilical cord region. ADSCs were isolated by enzymatic digestion method followed by enriching through a 41 μm filter. Filtered cells were then resuspended in cell culture flasks containing growth enriching medium and cultured in 5% CO2 in air at 37°C for 5 days. ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD44, CD90, and CD166 and by immunocytochemistry of mesenchymal stem cell specific protein i.e. CD44 and CD90. ADSCs were further in vitro differentiated. ADSCs derived osteocytes, chondrocytes, and adipocytes were validated through the amplification of specific markers of osteocytes (Osteopontin, Collagen I); chondrocytes (Aggrecan and Collagen II) and adipocytes (LPL, PPARα, PPARγ). Dog ADSCs were further autogenic transplanted into hip dysplasia and paraplegic patients. These patients recovered well one month from transplantation and were able to move freely. It may be concluded that these findings may have implications for defining the physiological roles of ADSCs in arthritis; orthopaedic ailments, joint regeneration, neuronal disorders, and several other applications leading to novel therapeutic opportunities.

198 ISOLATION, CHARACTERIZATION, AND IN VITRO DIFFERENTIATION OF GOAT ADIPOSE-TISSUE-DERIVED MESENCHYMAL STEM CELLS INTO PANCREATIC ISLETS-LIKE CELLS

The present study was carried out for isolation of goat (Capra hircus) adipose-tissue-derived stem cells (gADSCs) from adipose tissue, their characterization, and in vitro differentiation of gADSCs into pancreatic islets-like cells by giving conditioned medium. Goat ADSCs were isolated from goat adipose tissue by the enzymatic digestion method and were enriched by filtering through a 41-μm filter. Thus, filtered cells resuspended in a cell culture flask containing growth enriching medium and cultured in 5% CO2 in air at 38.5°C. Goat ADSCs were characterised by amplification of mesenchymal stem cell specific markers i.e. CD29, CD34, CD44, CD90, and CD166 as positive markers and CD41 and CD71 as negative markers. Immunocytochemistry of mesenchymal stem cell was also carried out with specific markers CD44 and CD90. Goat ADSCs were further characterised by in vitro differentiating them into osteocytes, chondrocytes, and adipocytes. For in vitro differentiation of gADSCs into osteocytes gADSCs were supplemented with conditioned medium i.e. DMEM containing fetal bovine serum (FBS), dexamethazone, B-glycerol phosphate and L-ascorbic acid. Osteogenic differentiation was confirmed by positive Alizarin red S staining and amplification of Osteopontin and Collagen I genes. For differentiation into chondrocytes cells, gADSCs were incubated in DMEM/F12 containing dexamethazone, ITX, BMP-4, and FBS for 21 days. Differentiated cells were confirmed by positive Safranin O staining and expression of chondrocytes specific Collagen III and Aggrecan genes. For adipogenesis, gADSCs were incubated with DMEM/F12 containing FBS, dexamethasone, and ITX and differentiated cells were confirmed by positive Oil Red O staining and amplification of adipocytes specific genes i.e. LPL, PPRγ and PPRα. For in-vitro differentiation gADSCs into pancreatic islets-like cells on the third or fourth passage gADSCs were incubated in conditioned medium containing serum-free DMEM/F12 medium with glucose (17.5 mM) in the presence of nicotinamide (10 mM), activin-A (2 nM), exendin-4 (10 nM), pentagastrin (10 nM), retinoic acid (10 μM) and mercaptoethanol (20 μM). The in vitro differentiation gADSCs into pancreatic islets-like cells was confirmed by amplification of pancreatic endoderm specific genes i.e. igf-1, sst, ngn3, pdx-1, isl-1, c-kit, thy-1, and Glut-2, and no expression was detected for above endoderm specific genes in undifferentiated gADSCs. Pancreatic islets-like cells were further characterised by immunostaining and Western blotting of Pdx-1, insulin, and Islets-1 specific protein. It could be concluded that gADSCs was differentiated into different lineages and secretory insulin was produced from pancreatic islets-like cells.

Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes

Mesenchymal stem cells (MSCs) are multipotent, can be easily expanded in culture and hence are an attractive therapeutic tool for cardiac repair. MSCs have tremendous potential to transdifferentiate to cardiac lineage both in vitro and in vivo. The present study examined the differentiation capacity of conditioned media derived from ischemic cardiac tissue on human MSCs. Human Bone marrow-derived MSCs after due characterization by immunocytochemistry and flow cytometry for MSC specific markers were induced by culture media derived from ischemic (n=13) and non-ischemic (n=18) human cardiac tissue. Parallel cultures were treated with 5-azacytidine (5-azaC), a potent cardiomyogen. MSCs induced with ischemic conditioned media formed myotube like structures, expressed sarcomeric Troponin I, alpha myosin heavy chain proteins and were positive for cardiac specific markers (Nkx2.5, human atrial natriuretic peptide, myosin light chain-2a, GATA-4) as was observed in 5-azaC treated cells. However, uninduced MSCs as well as those induced with non-ischemic cardiac conditioned media still maintained the fibroblast morphology even after 3weeks post-induction. Transmission electron microscopic studies of cardiomyocyte-like cells derived from MSCs revealed presence of sarcomeric bands but failed to show gap junctions and intercalated discs as of adult cardiomyocytes. These findings demonstrate that ischemic cardiac conditioned media induces morphological and molecular changes in MSCs with cardiac features, but at a primitive stage. Proteomics analysis of the ischemic conditioned media revealed differential expression of three relevant proteins (C-type lectin superfamily member 13, Testis-specific chromodomain protein Y2 and ADP/ATP translocase 1), whose exact role in cardiac regeneration needs further analysis.

The MET oncogene transforms human primary bone-derived cells into osteosarcomas by targeting committed osteo-progenitors

The MET oncogene is aberrantly overexpressed in human osteosarcomas. We have previously converted primary cultures of human bone-derived cells into osteosarcoma cells by overexpressing MET. To determine whether MET transforms mesenchymal stem cells or committed progenitor cells, here we characterize distinct MET overexpressing osteosarcoma (MET-OS) clones using genome-wide expression profiling, cytometric analysis, and functional assays. All the MET-OS clones consistently display mesenchymal and stemness markers, but not most of the mesenchymal–stem cell-specific markers. Conversely, the MET-OS clones express genes characteristic of early osteoblastic differentiation phases, but not those of late phases. Profiling of mesenchymal stem cells induced to differentiate along osteoblast, adipocyte, and chondrocyte lineages confirms that MET-OS cells are similar to cells at an initial phase of osteoblastic differentiation. Accordingly, MET-OS cells cannot differentiate into adipocytes or chondrocytes, but can partially differentiate into osteogenic-matrix-producing cells. Moreover, in vitro MET-OS cells form self-renewing spheres enriched in cells that can initiate tumors in vivo. MET kinase inhibition abrogates the self-renewal capacity of MET-OS cells and allows them to progress toward osteoblastic differentiation. These data show that MET initiates the transformation of a cell population that has features of osteo-progenitors and suggest that MET regulates self-renewal and lineage differentiation of osteosarcoma cells.

210 COMPARATIVE CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM MINIATURE PIG SYNOVIUM, SYNOVIAL FLUID AND BONE MARROW

Mesenchymal stem cells (MSC) are a valuable cell source for cartilage tissue engineering because MSC derived from various tissues readily differentiate into skeletal cell and chondrogenic lineages. In this study, we compared the cellular characteristics of synovium (SN) and synovial fluid (SF)-derived MSC with bone marrow (BM)-derived MSC of miniature pig. Biopsies of SN and BM were collected and SF was flushed with saline solution from 6-month-old miniature pig (T-type, PWG Micro-pig, PWG Genetics Korea, South Korea). Cells were isolated from collected tissues and cultured in advanced-DMEM supplemented with 10% fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2 in air. The cells were then evaluated for their expression of MSC-specific markers, including CD29, CD44 and CD90 using flow cytometry. The expression of early transcriptional factors, such as Oct3/4, Nanog and Sox2 was evaluated by immunocytochemical staining and reverse transcription-PCR (RT-PCR). Multilineage differentiation of MSC were induced under conditions conductive for osteogenic, adipogenic and chondrogenic lineages and then evaluated by von Kossa and Alizarin Red S staining, Oil red O staining and Alcian Blue staining, respectively. Differentiated cells were further analysed for the expression of lineage specific markers by RT-PCR. Statistical analysis was performed using one-way ANOVA by SPSS. The SN-, SF- and BM-MSC were observed to be positive for MSC specific markers, such as CD29 (99 ± 0.2, 96 ± 0.5 and 98 ± 0.2, respectively), CD44 (97 ± 0.3, 96 ± 0.6 and 98 ± 0.5, respectively) and CD90 (95 ± 0.5, 92 ± 0.2 and 96 ± 0.3, respectively); however, haematopoietic marker CD45 (2.0 ± 2.1, 3.0 ± 1.3 and 2.0 ± 3.0, respectively) was barely detected. In all MSC, early transcription factors (Oct3/4, Nanog and Sox2) were expressed by immnocytochemical staining and the transcripts were detected by RT-PCR. Following exposure to the specific differentiation medium, all these MSC were capable of differentiating into mesenchymal lineages, such as osteogenic, adiopogenic and chondrogenic as assessed by von Kossa and Alizarin Red S staining, Oil red O staining and and Alcian Blue staining, respectively. In addition, differentiated cells from all MSC expressed the marker genes specific to osteocytes (osteonectin, Runx2), adipocytes (aP2, PRAR-γ2) and chondrocytes (aggrecan, collagen type 2) by RT-PCR. The results of this study suggested that cells isolated from miniature pig articular tissues, such as SN and SF have similar characteristics of MSCs and their differentiation ability was comparable to BM-MSC. Hence, it is possible to establish MSCs from SN and SF as alternate sources during the biopsy of synovial tissues for arthritis diagnosis. Further studies are being carried out to evaluate their in vivo differentiation potential into chondrocytes. This study was supported by Rural Development Administration (grant No. 20110701-305-533-001-02-00) and National Research Foundation of Korea (grant No. 2011-0010252) of the Republic of Korea.

212 PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS DIFFERENTIATE IN VITRO INTO SMOOTH MUSCLE CELLS.

In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. This work was supported by Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (2010-0010528) and BioGreen 21 (20070301034040), Republic of Korea.

Size-sieved subpopulations of mesenchymal stem cells from intervascular and perivascular equine umbilical cord matrix

Umbilical cord matrix (UCM) has been recently proposed as an alternative source of mesenchymal stem cells (MSCs). The aim of this study was to isolate and characterize presumptive stem cells from intervascular and perivascular equine UCM and to obtain homogeneous subpopulations from both sites. Umbilical cords were processed for retrieval of MSCs. Unsieved cells from intervascular and perivascular portions were evaluated for cell cycle analysis and for immunophenotyping by flow cytometry. Cells from each site were separated into larger and smaller sieved populations using multi-dishes with 8-μm pore transwell inserts. Each cell population was characterized in terms of renewal capability, specific marker expression and differentiation potential. Cryopreservation was performed on sieved cells only. Cells from both areas expressed MSC and pluripotential specific markers and were able to differentiate into mesodermic and ectodermic lineages. The sieving procedure yielded two relatively homogeneous subpopulations with comparable characteristics. Surprisingly, after sieving, large intervascular and small perivascular cells were the most rapidly replicating cells [20.53 and 19.49 cell population doublings (PD) after 31 days respectively] and also showed higher fibroblast colony forming unit frequency. Unsieved cell populations were used as controls, and showed PD of 9.42(intervascular cells) and 8.54 (perivascular cells) after 31 days. Here, cells from UCM represented an intermediate stage between pluripotent embryonic and adult stem cells. Size-sieving can be used to isolate more rapidly proliferating cell populations.

Comparison of ganglioside expression between human adipose- and dental pulp-derived stem cell differentiation into osteoblasts

Human adipose-derived stem cells (hADSCs) and dental pulp-derived stem cells (hDPSCs) have been considered alternative sources of adult stem cells because of their potential to trans-differentiate into multiple cell lineages. This study investigated the possible role of gangliosides in the osteoblast differentiation of hADSCs and hDPSCs. First, we investigated characterization of hADSCs and hDPSCs using FACS analysis. Mesenchymal stem cell specific markers, CD44 and CD105, were expressed but not hematopoietic markers, CD45 and CD117 in both of hADSCs and hDPSCs. High-performance thin-layer chromatography analysis showed that increased gangliosides were associated with differentiation of hADSCs and hDPSCs into osteoblasts. RT-PCR analysis confirmed that osteoblast specific genes, ALP, BMP-2, collagen were expressed in differentiated osteoblasts, however, the another osteoblast specific gene, osteocalcin, was not expressed. When hADSCs and hDPSCs were cultured under osteoblast-differentiation conditions, alkaline phosphatase (ALP) activity was increased in comparison to hADSCs and hDPSCs. Furthermore, specifically both ALP activity and ganglioside expression increased more in hDPSCs-derived osteoblasts than hADSCs-derived osteoblasts. These results suggest that gangliosides play a more important role in regulating the osteoblast-differentiation of hDPSCs compared to hADSCs.