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Adipose derived stromal cells encapsulation in hydrogel particles: potential application to osteoarthritis
S508
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
release of the cells from the gel (by enzymatic digestion) at the end of their chondrogenic conversion, giving therefore the possibility to analyze them directly by flow cytometry. Recently, we developed and characterized the first antibody capable of detecting the IIB isoform of human type II procollagen, the only isoform of this collagen type that is expressed by well-differentiated chondrocytes. Here, we used this antibody by flow cytometry to look for intracellular IIB procollagen expression in MSCs undergoing chondrocyte differentiation in hydrogel. Results: The mean population doubling times between P1 and P5 ranged between 30 h and 55 h, depending on the tissue origin of MSCs. The doubling times remained constant from P1 to P5 and a minimal reservoir of 300 10^6 cells (e.g., BM-MSCs) could be obtained after 5 passages, starting with 1 10^6 cells harvested at the end of P1. Importantly, no karyotype abnormalities were detected after 5 passages. In brief, all cell sources yielded clinical-scale amounts of MSCs after expansion in serum-free culture conditions. By using an original panel of 27 surface markers that were analyzed by 8-color flow cytometry, our results revealed that all MSC sources were positive for the expression of the ISCT-validated markers (CD73, CD90, CD105) as well as for other recognized MSC markers (CD44). Nevertheless, differences in expression for other markers were found between the sources. BM-MSCs were the only cells to include subpopulations positive for all the putative skeletal markers analyzed (CD29, CD56, MSCA1, Stro-1, CD106, CD146, CD271). All cell sources showed signs of adipogenic, osteogenic and chondrogenic conversion in monolayer cultures, after amplification in serum-free conditions. However, only BMMSCs were able to clearly convert into true chondrocytes in micromass pellets or hydrogel cultures, as attested in particular by the expression of type IIB procollagen, monitored by immunohistochemistry or flow cytometry. Conclusions: It is possible to drive MSCs to the chondrogenic lineage in hydrogel after expansion and differentiation in serum-free conditions. BM-MSCs appear to be the best candidates for chondrogenic conversion in the conditions used in this study and this is in concordance with the largest panel of putative skeletal markers revealed by flow cytometry analysis for this category of MSCs. In this context, we have introduced an innovative quality control of the chondrocytic status of MSCs embedded in hydrogel, via measurement by flow cytometry of intracellular IIB procollagen, a marker of true chondrocytes. 874 DIFFERENTIAL CHARACTERISTICS BETWEEN CARTILAGE AND BONE MARROW MESENCHYMAL STEM CELLS IN OSTEOARTHRITIC HUMAN KNEES W.-S. Lian y, J.-Y. Ko z, F.-S. Wang y, x. y Core Lab. for Phenomics and Diagnostics, Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Taiwan; z Dept. of Orthopedic Surgery, Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Kaohsiung, Taiwan; x Ctr. for Translational Res. in BioMed. Sci., Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Taiwan Purpose: Osteoarthritis (OA) of the knee is accounted for the leading cause of joint pain, disability and total knee arthroplasty. Severe articular cartilage loss, synovial inflammation and subchondral microenvironment damage are prominent features of OA. Mesenchymal stem cells (MSCs) reportedly have multiple biological capacities, including chondrogenic lineage specification, extracellular matrix synthesis and immune modulation through TGF-b, Smad and BMP signaling in the development and remodeling of joint tissue. This study was undertaken to characterize and comparative exhibit profiles on the stemness and lineage specification of cartilage MSCs (CMSCs) with bone marrow MSCs (BMMSCs) from OA patients. Methods: This study was approved by IRB (no. 104-5248B). Informed consent was obtained from all participants. Cartilage tissue and bonemarrow in 40 patients with end stage OA knees underwent total knee replacement were harvested for isolating CMSCs and BMMSCs, respectively. For the collection of CMSCs, The remaining specimens were incubated in a-MEM contain 20% FBS culture medium for 7 days and followed by harvested interesting migrating out of cells. Proliferation and surface antigen expression of CMSCs and BMMSCs were assessed by cell counts and flow cytometry. MSCs were incubated in osteogenic, chondrogenic, and adipogenic conditions to assess lineage commitment. All experiments were performed within 2e3 passages of CMSCs and BMMSCs. Data were presented as mean ± S.D. Two groups were compared using the Student's t-test. Statistical analysis was
performed with one-way ANOVA. The differences were considered significant at a value of P < 0.05. Results: The majority of CMSCs exhibited as tight spindle-shaped, whereas BMMSCs predominantly showed a flat spindle-like morphology. Surface antigen expression of MSCs was varied between CMSCs and BMMSCs. For example, CMSCs had lower expression of CD105 (93% vs 98%), CD73 (18.6% vs 66%), CD49f (20% vs 33%) and CD166 (0.6% vs 19,2%) and higher CD90 expression (98% v.s 89%) compared to those of BMMSCs. Furthermore, CMSCs had increased proliferation capacity than BMMSCs. It is noteworthy that CMSCs exhibited increased activation of osteogenic differentiation and lessened adipogenic lineage commitment. The CMSCs had evaluated and significant expression of embryonic markers OCT3/4 and concomitant with a mitigated expression of inflammatory, fibroblast regulators and angiogenesis, including IL-1b, TNF-a, TGF-b, SMAD3, THPS1 and GPR27 as compared to BMMSCs by Q-PCR analysis. Additionally, the influence of extracellular matrix factors was further analyzed and observation elevated in collagen II and HMGB1 protein expression on CMSCs. Conclusions: Taken together, growth and stem cell phenotypes of MSCs in OA knees depend on joint micro-compartment. CMSCs have increased self-renewal ability, surface markers expression and bone formation capacity. Increased embryonic markers and weakened inflammatory and fibroblast regulator expression of CMSCs explains the phenomenon that a niche favorable to CMSCs growth and stemness existed in joint lesions. This study opens a new door into understanding the pathogenesis of OA and regeneration potential for OA joint repair. 875 ADIPOSE DERIVED STROMAL CELLS ENCAPSULATION IN HYDROGEL PARTICLES: POTENTIAL APPLICATION TO OSTEOARTHRITIS F. Hached y, z, C. Vinatier y, z, P.-G. Pinta y, z, P. Weiss y, z, C. Le Visage y, z, P. Hulin x, A. Billon-Chabaud y, k, J. Guicheux y, z, G. Grimandi y, k. y LIOAD Inserm U791, Nantes, France; z Faculty of Dentistry, Nantes, France; x SFR François Bonamy, Plateforme MicroPicell, Inserm UMS 016, Nantes, France; k Faculty of Pharmacy, Nantes, France Purpose: Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects cartilage, subchondral bone and joint tissues. Mesenchymal Stromal Cells (MSCs) have generated interest since they secrete immuno-modulatory and anti-inflammatory factors. Since intra-articular injection of MSCs suffers major limitations including a cell death upon injection and a massive leakeage outside the articular space, we proposed in this study to entrap MSCs within particles derived from alginate or silylated hydroxypropyl methylcellulose (SiHPMC) hydrogels. Methods: MSCs were isolated from human adipose tissue. We used a dropwise method in CaCl2 solution and developed a water/oil emulsion protocol to obtain alginate and Si-HPMC particles, respectively. To assess the hydrogel network, particles were incubated in FITC-dextran (Mw 20e2000 kDa) solutions for 2 h. Diffusion profiles were assessed by confocal microscopy. Ratio of internal to external fluorescence was calculated (a ratio of 1 indicates that equilibrium is reached). Mechanical properties of the particles were investigated by subjecting them to a 30% compressive displacement for 30 s (MicroSquisher). A suspension of 2.106 MSCs/ml was added either to the alginate or SiHPMC solution, the particles were collected and cultured for up to 2 months. Cell viability was followed using a Live/Dead assay. To assess their anti-inflammatory properties, MSCs were treated for 72 hours with pro-inflammatory molecules (TNF-a, IFN-y) in the culture medium. Release of prostaglandin E2 (PGE2) was measured using a EIA Kit (Cayman) and indoleamine 2,3-dioxygenase (IDO) activity was measured by tryptophan-to-kynurenine conversion. Results: We obtained alginate and Si-HPMC particles with an average size of 1.5 ± 0.2 mm and 75 ± 28 mm, respectively. We evidenced a faster diffusion in Si-HPMC particles than in alginate ones (Figure 1). Equilibrium was reached after 2 hours for 20 kDa dextran in Si-HPMC, with less than 30% for alginate. This data suggests that Si-HPMC could not only favor cell stimulation by the inflammatory signals but also support the release of secreted molecules. Under compression, alginate particles exhibited a Young's modulus that was 70 times greater than Si-HPMC ones (Figure 2), thereby suggesting that viscoelastic Si-HPMC particles could be more effective at protecting cells from shear stress during injection and in the articular cavity. Both Si-HPMC and alginate particles supported cell survival (viability at 2 months of 93% and 90%, respectively; Figure 3). The stimulation of encapsulated MSCs resulted in a 3-
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
fold increase in PGE2 production in the supernatant after stimulation with pro-inflammatory cytokines, while IDO activity increased 33 times as compared to non-stimulated cells. Conclusions: We have demonstrated that Si-HPMC and alginate particles support MSCs viability and the maintenance of their antiinflammatory properties. Diffusion assays and mechanical properties experiments indicated that Si-HPMC is a better candidate for MSCs encapsulation than alginate. Future experiments will investigate whether encapsulated MSCs in Si-HPMC may be a relevant strategy to prevent cartilage degradation and inflammation in OA.
Figure 1: Assessment of Si-HPMC and alginate particle diffusion properties.
Figure 2: Mechanical properties of the particles after subjecting them to a 30% compressive displacement for 30 s (MicroSquisher).
S509
Purpose: Osteoarthritis (OA) is a progressive degenerative disorder affecting several compartments of the joints, including articular cartilage. Cell therapy with chondrocytes or mesenchymal stem cells (MSCs) to repair focal lesions of the joint surface, a known risk factor for OA, has been intensely pursued. Although studies have demonstrated improvements in symptomatic outcome and cartilage repair, patientto-patient variability in structural outcome has been reported. To enhance consistency, cell therapy requires a better insight into MSC signature molecules to select qualifying donors and compliant cell sources for articular cartilage repair with MSCs. In this study we aimed to analyse human MSC transcriptomic and proteomic profiles to identify tissue-specific molecular signatures and biomarkers predictive of their chondrogenic potency. Methods: Human MSCs from three different joint tissues (human bone marrow ¼ HBM, human periosteum ¼ HP, human synovial membrane ¼ HSM) were culture-expanded. In vitro chondrogenesis assays were performed to select samples with high or low chondrogenic differentiation capacity based on their COL2A1/COL1A1 ratio. Microarrays and liquid chromatography-mass spectrometry (LC-MS) were carried out to compare the transcriptome and proteome profiles of MSCs with high vs. low chondrogenic potency and across the three different tissue sources. Expression of selected candidate genes/proteins was validated by q-RT-PCR and Western blot. Results: Analysis of the microarray and proteomic datasets revealed significant differences in gene expression profiles between MSCs from high and low chondrogenic potency groups and also across the three tissue sources. The high potency group had highly enriched signature molecules with many important biological functions (e.g., gene expression, protein translation, protein phosphorylation, protein transportation, cell division, cytoskeleton organization, intracellular signal transduction etc.). HSM and BM-MSCs expressed distinct sets of genes. A total of 347 genes and 47 pathways, including pathways involved in skeletogenesis, were differentially expressed. Western blot and proteomic analysis confirmed a unique tissue-specific molecular signature for HSM-MSCs. Conclusions: Our findings demonstrate that MSCs with relatively high chondrogenic differentiation capacity exhibit a distinct genomic and proteomic profile. We have also demonstrated that HSM and BM-MSCs express distinct sets of genes. Gene lists could be potential molecular signatures for tissue-specific MSC identity in clinical applications. 877 DIFFERENT METHODS OF DETACHING ADHERENT CELLS SIGNIFICANTLY AFFECT THE DETECTION OF STEM CELL ANTIGENS IN SYNOVIAL MESENCHYMAL STEM CELLS M. Ojima y, K. Tsuji z, K. Otabe x, M. Horie x, H. Koga y, I. Sekiya x, T. Muneta y. y Dept. of Joint Surgery and Sports Med., Tokyo Med. and Dental Univ., Tokyo, Japan; z Dept. of Cartilage Regeneration, Tokyo Med. and Dental Univ., Tokyo, Japan; x Ctr. for Stem Cell and Regenerative Med., Tokyo Med. and Dental Univ., Tokyo, Japan
Figure 3. MSCs viability after encapsulation in Si-HPMC and alginate particles. The viability was performed using the UltraVIEW VoX 3D Live Cell Imaging System with Volocity Software. 876 TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF SIGNATURE MOLECULES PREDICTIVE OF CHRONDROGENIC POTENCY AND TISSUE SPECIFICITY IN HUMAN MESENCHYMAL STEM CELLS S. Shahana, A. Roelofs, C. De Bari. Aberdeen Univ., Aberdeen, United Kingdom
Purpose: Cultured fibroblastic cells isolated from supra-patella synovial membrane in the knee joint have characteristics of mesenchymal stem cells (MSCs) since they are plastic-adherent cells when maintained in standard culture conditions, express CD73, CD90, CD105 surface antigens, and have ability to differentiate to osteoblasts, adipocytes, and chondroblasts in vitro. We have reported that intra-articular administration of synovial MSC suspension significantly enhanced both articular cartilage and meniscus regeneration in experimental animals such as rats, rabbits, and microminipigs. Based on these results, we have started clinical trials for articular cartilage regeneration by the administration of autologous synovial MSC suspensions. In these clinical trials, MSCs are isolated from autologous tissues obtained by synovial biopsies and expanded in our University Hospital’s Cell Processing Center (ISO9001 certified). To establish the effective and reproducible MSC transplantation therapy, we consider it quite important to prepare cell suspension with minimum damage on MSCs. In this study, we examined the effects of different methods of detaching adherent cells (enzymatic and non-enzymatic) on the viability and expression of stem cell antigens in cultured synovial MSCs. Methods: This study was approved by the Ethics Committee of Tokyo Medical and Dental University and informed consent was obtained from all subjects. Primary human synovial MSCs, those were prepared from synovial membrane obtained from patients who underwent total knee arthroplasty (TKA), were cultured for 14 days in MEM-alpha
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
release of the cells from the gel (by enzymatic digestion) at the end of their chondrogenic conversion, giving therefore the possibility to analyze them directly by flow cytometry. Recently, we developed and characterized the first antibody capable of detecting the IIB isoform of human type II procollagen, the only isoform of this collagen type that is expressed by well-differentiated chondrocytes. Here, we used this antibody by flow cytometry to look for intracellular IIB procollagen expression in MSCs undergoing chondrocyte differentiation in hydrogel. Results: The mean population doubling times between P1 and P5 ranged between 30 h and 55 h, depending on the tissue origin of MSCs. The doubling times remained constant from P1 to P5 and a minimal reservoir of 300 10^6 cells (e.g., BM-MSCs) could be obtained after 5 passages, starting with 1 10^6 cells harvested at the end of P1. Importantly, no karyotype abnormalities were detected after 5 passages. In brief, all cell sources yielded clinical-scale amounts of MSCs after expansion in serum-free culture conditions. By using an original panel of 27 surface markers that were analyzed by 8-color flow cytometry, our results revealed that all MSC sources were positive for the expression of the ISCT-validated markers (CD73, CD90, CD105) as well as for other recognized MSC markers (CD44). Nevertheless, differences in expression for other markers were found between the sources. BM-MSCs were the only cells to include subpopulations positive for all the putative skeletal markers analyzed (CD29, CD56, MSCA1, Stro-1, CD106, CD146, CD271). All cell sources showed signs of adipogenic, osteogenic and chondrogenic conversion in monolayer cultures, after amplification in serum-free conditions. However, only BMMSCs were able to clearly convert into true chondrocytes in micromass pellets or hydrogel cultures, as attested in particular by the expression of type IIB procollagen, monitored by immunohistochemistry or flow cytometry. Conclusions: It is possible to drive MSCs to the chondrogenic lineage in hydrogel after expansion and differentiation in serum-free conditions. BM-MSCs appear to be the best candidates for chondrogenic conversion in the conditions used in this study and this is in concordance with the largest panel of putative skeletal markers revealed by flow cytometry analysis for this category of MSCs. In this context, we have introduced an innovative quality control of the chondrocytic status of MSCs embedded in hydrogel, via measurement by flow cytometry of intracellular IIB procollagen, a marker of true chondrocytes. 874 DIFFERENTIAL CHARACTERISTICS BETWEEN CARTILAGE AND BONE MARROW MESENCHYMAL STEM CELLS IN OSTEOARTHRITIC HUMAN KNEES W.-S. Lian y, J.-Y. Ko z, F.-S. Wang y, x. y Core Lab. for Phenomics and Diagnostics, Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Taiwan; z Dept. of Orthopedic Surgery, Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Kaohsiung, Taiwan; x Ctr. for Translational Res. in BioMed. Sci., Kaohsiung Chang Gung Mem. Hosp., Kaohsiung, Taiwan Purpose: Osteoarthritis (OA) of the knee is accounted for the leading cause of joint pain, disability and total knee arthroplasty. Severe articular cartilage loss, synovial inflammation and subchondral microenvironment damage are prominent features of OA. Mesenchymal stem cells (MSCs) reportedly have multiple biological capacities, including chondrogenic lineage specification, extracellular matrix synthesis and immune modulation through TGF-b, Smad and BMP signaling in the development and remodeling of joint tissue. This study was undertaken to characterize and comparative exhibit profiles on the stemness and lineage specification of cartilage MSCs (CMSCs) with bone marrow MSCs (BMMSCs) from OA patients. Methods: This study was approved by IRB (no. 104-5248B). Informed consent was obtained from all participants. Cartilage tissue and bonemarrow in 40 patients with end stage OA knees underwent total knee replacement were harvested for isolating CMSCs and BMMSCs, respectively. For the collection of CMSCs, The remaining specimens were incubated in a-MEM contain 20% FBS culture medium for 7 days and followed by harvested interesting migrating out of cells. Proliferation and surface antigen expression of CMSCs and BMMSCs were assessed by cell counts and flow cytometry. MSCs were incubated in osteogenic, chondrogenic, and adipogenic conditions to assess lineage commitment. All experiments were performed within 2e3 passages of CMSCs and BMMSCs. Data were presented as mean ± S.D. Two groups were compared using the Student's t-test. Statistical analysis was
performed with one-way ANOVA. The differences were considered significant at a value of P < 0.05. Results: The majority of CMSCs exhibited as tight spindle-shaped, whereas BMMSCs predominantly showed a flat spindle-like morphology. Surface antigen expression of MSCs was varied between CMSCs and BMMSCs. For example, CMSCs had lower expression of CD105 (93% vs 98%), CD73 (18.6% vs 66%), CD49f (20% vs 33%) and CD166 (0.6% vs 19,2%) and higher CD90 expression (98% v.s 89%) compared to those of BMMSCs. Furthermore, CMSCs had increased proliferation capacity than BMMSCs. It is noteworthy that CMSCs exhibited increased activation of osteogenic differentiation and lessened adipogenic lineage commitment. The CMSCs had evaluated and significant expression of embryonic markers OCT3/4 and concomitant with a mitigated expression of inflammatory, fibroblast regulators and angiogenesis, including IL-1b, TNF-a, TGF-b, SMAD3, THPS1 and GPR27 as compared to BMMSCs by Q-PCR analysis. Additionally, the influence of extracellular matrix factors was further analyzed and observation elevated in collagen II and HMGB1 protein expression on CMSCs. Conclusions: Taken together, growth and stem cell phenotypes of MSCs in OA knees depend on joint micro-compartment. CMSCs have increased self-renewal ability, surface markers expression and bone formation capacity. Increased embryonic markers and weakened inflammatory and fibroblast regulator expression of CMSCs explains the phenomenon that a niche favorable to CMSCs growth and stemness existed in joint lesions. This study opens a new door into understanding the pathogenesis of OA and regeneration potential for OA joint repair. 875 ADIPOSE DERIVED STROMAL CELLS ENCAPSULATION IN HYDROGEL PARTICLES: POTENTIAL APPLICATION TO OSTEOARTHRITIS F. Hached y, z, C. Vinatier y, z, P.-G. Pinta y, z, P. Weiss y, z, C. Le Visage y, z, P. Hulin x, A. Billon-Chabaud y, k, J. Guicheux y, z, G. Grimandi y, k. y LIOAD Inserm U791, Nantes, France; z Faculty of Dentistry, Nantes, France; x SFR François Bonamy, Plateforme MicroPicell, Inserm UMS 016, Nantes, France; k Faculty of Pharmacy, Nantes, France Purpose: Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects cartilage, subchondral bone and joint tissues. Mesenchymal Stromal Cells (MSCs) have generated interest since they secrete immuno-modulatory and anti-inflammatory factors. Since intra-articular injection of MSCs suffers major limitations including a cell death upon injection and a massive leakeage outside the articular space, we proposed in this study to entrap MSCs within particles derived from alginate or silylated hydroxypropyl methylcellulose (SiHPMC) hydrogels. Methods: MSCs were isolated from human adipose tissue. We used a dropwise method in CaCl2 solution and developed a water/oil emulsion protocol to obtain alginate and Si-HPMC particles, respectively. To assess the hydrogel network, particles were incubated in FITC-dextran (Mw 20e2000 kDa) solutions for 2 h. Diffusion profiles were assessed by confocal microscopy. Ratio of internal to external fluorescence was calculated (a ratio of 1 indicates that equilibrium is reached). Mechanical properties of the particles were investigated by subjecting them to a 30% compressive displacement for 30 s (MicroSquisher). A suspension of 2.106 MSCs/ml was added either to the alginate or SiHPMC solution, the particles were collected and cultured for up to 2 months. Cell viability was followed using a Live/Dead assay. To assess their anti-inflammatory properties, MSCs were treated for 72 hours with pro-inflammatory molecules (TNF-a, IFN-y) in the culture medium. Release of prostaglandin E2 (PGE2) was measured using a EIA Kit (Cayman) and indoleamine 2,3-dioxygenase (IDO) activity was measured by tryptophan-to-kynurenine conversion. Results: We obtained alginate and Si-HPMC particles with an average size of 1.5 ± 0.2 mm and 75 ± 28 mm, respectively. We evidenced a faster diffusion in Si-HPMC particles than in alginate ones (Figure 1). Equilibrium was reached after 2 hours for 20 kDa dextran in Si-HPMC, with less than 30% for alginate. This data suggests that Si-HPMC could not only favor cell stimulation by the inflammatory signals but also support the release of secreted molecules. Under compression, alginate particles exhibited a Young's modulus that was 70 times greater than Si-HPMC ones (Figure 2), thereby suggesting that viscoelastic Si-HPMC particles could be more effective at protecting cells from shear stress during injection and in the articular cavity. Both Si-HPMC and alginate particles supported cell survival (viability at 2 months of 93% and 90%, respectively; Figure 3). The stimulation of encapsulated MSCs resulted in a 3-
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
fold increase in PGE2 production in the supernatant after stimulation with pro-inflammatory cytokines, while IDO activity increased 33 times as compared to non-stimulated cells. Conclusions: We have demonstrated that Si-HPMC and alginate particles support MSCs viability and the maintenance of their antiinflammatory properties. Diffusion assays and mechanical properties experiments indicated that Si-HPMC is a better candidate for MSCs encapsulation than alginate. Future experiments will investigate whether encapsulated MSCs in Si-HPMC may be a relevant strategy to prevent cartilage degradation and inflammation in OA.
Figure 1: Assessment of Si-HPMC and alginate particle diffusion properties.
Figure 2: Mechanical properties of the particles after subjecting them to a 30% compressive displacement for 30 s (MicroSquisher).
S509
Purpose: Osteoarthritis (OA) is a progressive degenerative disorder affecting several compartments of the joints, including articular cartilage. Cell therapy with chondrocytes or mesenchymal stem cells (MSCs) to repair focal lesions of the joint surface, a known risk factor for OA, has been intensely pursued. Although studies have demonstrated improvements in symptomatic outcome and cartilage repair, patientto-patient variability in structural outcome has been reported. To enhance consistency, cell therapy requires a better insight into MSC signature molecules to select qualifying donors and compliant cell sources for articular cartilage repair with MSCs. In this study we aimed to analyse human MSC transcriptomic and proteomic profiles to identify tissue-specific molecular signatures and biomarkers predictive of their chondrogenic potency. Methods: Human MSCs from three different joint tissues (human bone marrow ¼ HBM, human periosteum ¼ HP, human synovial membrane ¼ HSM) were culture-expanded. In vitro chondrogenesis assays were performed to select samples with high or low chondrogenic differentiation capacity based on their COL2A1/COL1A1 ratio. Microarrays and liquid chromatography-mass spectrometry (LC-MS) were carried out to compare the transcriptome and proteome profiles of MSCs with high vs. low chondrogenic potency and across the three different tissue sources. Expression of selected candidate genes/proteins was validated by q-RT-PCR and Western blot. Results: Analysis of the microarray and proteomic datasets revealed significant differences in gene expression profiles between MSCs from high and low chondrogenic potency groups and also across the three tissue sources. The high potency group had highly enriched signature molecules with many important biological functions (e.g., gene expression, protein translation, protein phosphorylation, protein transportation, cell division, cytoskeleton organization, intracellular signal transduction etc.). HSM and BM-MSCs expressed distinct sets of genes. A total of 347 genes and 47 pathways, including pathways involved in skeletogenesis, were differentially expressed. Western blot and proteomic analysis confirmed a unique tissue-specific molecular signature for HSM-MSCs. Conclusions: Our findings demonstrate that MSCs with relatively high chondrogenic differentiation capacity exhibit a distinct genomic and proteomic profile. We have also demonstrated that HSM and BM-MSCs express distinct sets of genes. Gene lists could be potential molecular signatures for tissue-specific MSC identity in clinical applications. 877 DIFFERENT METHODS OF DETACHING ADHERENT CELLS SIGNIFICANTLY AFFECT THE DETECTION OF STEM CELL ANTIGENS IN SYNOVIAL MESENCHYMAL STEM CELLS M. Ojima y, K. Tsuji z, K. Otabe x, M. Horie x, H. Koga y, I. Sekiya x, T. Muneta y. y Dept. of Joint Surgery and Sports Med., Tokyo Med. and Dental Univ., Tokyo, Japan; z Dept. of Cartilage Regeneration, Tokyo Med. and Dental Univ., Tokyo, Japan; x Ctr. for Stem Cell and Regenerative Med., Tokyo Med. and Dental Univ., Tokyo, Japan
Figure 3. MSCs viability after encapsulation in Si-HPMC and alginate particles. The viability was performed using the UltraVIEW VoX 3D Live Cell Imaging System with Volocity Software. 876 TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF SIGNATURE MOLECULES PREDICTIVE OF CHRONDROGENIC POTENCY AND TISSUE SPECIFICITY IN HUMAN MESENCHYMAL STEM CELLS S. Shahana, A. Roelofs, C. De Bari. Aberdeen Univ., Aberdeen, United Kingdom
Purpose: Cultured fibroblastic cells isolated from supra-patella synovial membrane in the knee joint have characteristics of mesenchymal stem cells (MSCs) since they are plastic-adherent cells when maintained in standard culture conditions, express CD73, CD90, CD105 surface antigens, and have ability to differentiate to osteoblasts, adipocytes, and chondroblasts in vitro. We have reported that intra-articular administration of synovial MSC suspension significantly enhanced both articular cartilage and meniscus regeneration in experimental animals such as rats, rabbits, and microminipigs. Based on these results, we have started clinical trials for articular cartilage regeneration by the administration of autologous synovial MSC suspensions. In these clinical trials, MSCs are isolated from autologous tissues obtained by synovial biopsies and expanded in our University Hospital’s Cell Processing Center (ISO9001 certified). To establish the effective and reproducible MSC transplantation therapy, we consider it quite important to prepare cell suspension with minimum damage on MSCs. In this study, we examined the effects of different methods of detaching adherent cells (enzymatic and non-enzymatic) on the viability and expression of stem cell antigens in cultured synovial MSCs. Methods: This study was approved by the Ethics Committee of Tokyo Medical and Dental University and informed consent was obtained from all subjects. Primary human synovial MSCs, those were prepared from synovial membrane obtained from patients who underwent total knee arthroplasty (TKA), were cultured for 14 days in MEM-alpha