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Evaluation of stem cell populations isolated from adipose tissue using minimally manipulated techniques
S74
Poster Abstracts
251 HIGH THROUGHPUT CHONDROGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS FOR CARTILAGE TISSUE ENGINEERING YM Lin, J Fang, Y Lim, S Reuveny, S Oh Stem Cell Group, Bioprocessing Technology Institute, Singapore, Singapore, Singapore Human mesenchymal stem cells (hMSC) are a clinically relevant population that is increasingly being investigated for autologous and allogeneic stem cell therapies. They are relevant for many therapeutic applications as they can differentiate into 3 cell types, namely chondrocytes for cartilage generation, osteoblasts for bone development, and adipocytes for fat formation. Fetal hMSC (fhMSC) are attractive for cell therapy use as they display higher growth rate, enhanced plasticity and slower senescence rate than adult hMSC do. As cartilage tissue has poor self-regenerative ability, degeneration of cartilage in diseases like osteoarthritis presents limited options for repair and replacement. Thus, in this study, we focus on directing fhMSC to differentiate into chondrocytes for scalable cartilage tissue engineering. Here, we present a high throughput method of generating chondrogenic pellets in vitro with ultra-low attachment 96 well plates that are more spacesaving and cost effective for scale-up operations than conventional methods are. Our cytokine screen and dosage-dependent studies revealed that BMP2, when used at 50-100 ng/ml, is more effective in inducing the chondrogenic differentiation of fhMSC than TGFb1/3 are. These results are supported by functional output assays that measure glycosaminoglycan and type II collagen content, and gene expression analysis. Calcium quantification and histological staining further ascertained that BMP2 does not induce bone formation from fhMSC. In all, our results demonstrate that BMP2 induces effective chondrogenic differentiation of fhMSC, rather than bone differentiation. They also suggest that there are innate differences in hMSC derived from distinct sources to external signaling factors. Understanding how these differences are mediated would inform future work delving into scalable cartilage tissue engineering.
252 EVALUATION OF STEM CELL POPULATIONS ISOLATED FROM ADIPOSE TISSUE USING MINIMALLY MANIPULATED TECHNIQUES S Mehta3, F Silva2, V Vargas2, C Elabd2, C Crombie3, A Patel1 1 Cardiothoracic Surgery, University of Utah, Salt Lake City, Utah, United States, 2BioRestorative Therapies, Long Island, New York, United States, 3Plastic and Reconstructive Surgery, University of Utah, Salt Lake City, Utah, United States Rational: Autologous fat grafting is a highly used minimally manipulated technique in plastic and reconstructive surgery. Several fat-processing techniques have been described such as centrifugation, Telfa-rolling, blue-towel, sieve and decanting methods). Although studies have compared processing methods, no studies have evaluated the processing time, cost and stem cell potency of the different methods. Objective: Here we describe a study where we compare the processing time, cost, degree of cell viability and adipose derived stem cell (ADSC) potency of adipose tissue processed using several minimally manipulated fat-processing techniques adipose tissue. Methods and Results: Human adipose tissue was harvested and processed using centrifugation, Telfa-rolling, blue-towel, sieve and decanting methods. Processed tissue was placed into culture using ADSC cell culture methods to allow cells to grow. Cells were tested for doubling times, cell surface marker analysis and differentiation potential. Conclusions: All methods resulted in the isolation of adipose derived stem cells capable of undergoing adipo, osteo and chondrogenesis. Telfa-rolling and centrifuge methods resulted in the highest number of cells immediately after processing, and reached a cell density of 20 x106 within 6 days of tissue processing. Telfa-rolling is the most cost effective minimally manipulated method of processing adipose tissue that results in the highest ADSC isolation. This study supports larger clinical studies to evaluate the clinical benefits of using the Telfa-rolling method as the preferred minimally manipulated technique used in fat grafting.
253 ANALYSIS OF GENE EXPRESSIONS AND METABOLIC ACTIVITIES IN FETAL AND POSTNATAL HUMAN HEPATOCYTES FOR CLINICAL THERAPY R Gramignoli1, K Kannisto1, RC Srinivasan1, T Miki2, SC Strom1 1 Laboratory Medicine, Karolinska Institute, Stockholm, Sweden, 2Department of Biochemistry and Molecular Biology, University Southern California, Los Angeles, California, United States Fetal hepatocytes have been proposed for clinical hepatocyte transplants. However, to be useful, sufficient metabolic activities must be present. We assessed the viability and metabolic activities in isolated fetal human hepatocytes as compared to pediatric and adult hepatocytes, and we evaluated liver repopulation post-transplant in mice. Hepatocytes were isolated from 254 fetal livers, 11-24 weeks gestation and 229 post-natal tissues (3 months-85 years). Gene expression profile and functional tests (including Cytochrome P450 (CYP) activities, phase II-conjugation and ammonia metabolism) were assessed after isolation and long-term culture. Cells were transplanted to SCID/beige mice previously treated with retrorsine, and liver tissue analyzed after 1, 3 and 6 months for human DNA content (Alu sequences) and RNA levels of several liver genes. The viability of isolated fetal hepatocytes was significantly higher compared to adults (919% vs 7921%, p<0.0001). CYP1A, 2C9 and 3A4 activity increased with gestational age, but remained 1-6% of adult values. The fetal form of CYP3A (3A7) was expressed at high levels, and its activity decrease after birth. Phase II-conjugation and ammonia metabolism were null/low in fetal hepatocytes immediately after isolation, but increased to levels observed in mature hepatocytes in cells cultured for 5 days. DNA analysis and immunohistochemistry confirmed the presence of human hepatocytes in mouse liver. The average level of repopulation with fetal cells was 18-times higher than that observed with adult hepatocytes at 6 months post transplant. Once engrafted, the expression of several mature liver genes increased, however theynever exceeded 20% of normal adult levels. Fetal hepatocytes may be useful for clinical transplants; however they lack significant metabolic activities when isolated. Maturation of the cells may take longer than previously expected, and their activities need to be matched with patient requirements. 254 WILL NOT BE PRESENTED 255 CD271 ENRICHMENT OF BM-DERIVED MSCs ENHANCES THEIR OSTEOGENIC POTENCY S Müller, J Tang, L Nicholson, X Nong-Wang, JR De Havilland, A Dickinson Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom Mesenchymal stem cells (MSCs) are a promising candidate of regenerative medicine in treating bone destruction in osteoarthritis (OA). In vitro expansion of MSCs isolated via plastic adherence reduces their potential for osteogenic differentiation. CD271highCD45- MSCs, thought to be MSC progenitors, have been shown to be osteogenicly primed and may be better suited to bone regeneration. This study compared MSC enriched for CD271 (CD271-MSC) with non-enriched plastic adherent populations (PA-MSC) to address whether CD271-MSC have more potent bone forming capacity. From 5 samples of bone marrow derived mononuclear cells, samples pre and post CD271 enrichment were used to generate PA-MSCs and CD271-MSC respectively, which were subsequently cultured in parallel. At passages 2, 3 and 6 the samples were assessed for phenotypic profile by flow cytometry and differentiation potential following lineage induction and chemical staining. Osteogenic gene expression was measured using real-time PCR. CD271 enrichment resulted in an over 1000 fold increase in the frequency of CD271highCD45- cells, from 0.01% (0.003 SEM) in PA-MSCs to 11% (1.77 SEM) in CD271-MSCs. At all passages, each population expressed the standard MSC phenotype. CD271-MSCs had a slightly enhanced rate of population doubling, reaching 12 (0.13 SEM) population doublings by passage 6, while the PA-MSCs had undergone 11 (0.74 SEM) population doublings.
Poster Abstracts
251 HIGH THROUGHPUT CHONDROGENIC DIFFERENTIATION OF HUMAN MESENCHYMAL STEM CELLS FOR CARTILAGE TISSUE ENGINEERING YM Lin, J Fang, Y Lim, S Reuveny, S Oh Stem Cell Group, Bioprocessing Technology Institute, Singapore, Singapore, Singapore Human mesenchymal stem cells (hMSC) are a clinically relevant population that is increasingly being investigated for autologous and allogeneic stem cell therapies. They are relevant for many therapeutic applications as they can differentiate into 3 cell types, namely chondrocytes for cartilage generation, osteoblasts for bone development, and adipocytes for fat formation. Fetal hMSC (fhMSC) are attractive for cell therapy use as they display higher growth rate, enhanced plasticity and slower senescence rate than adult hMSC do. As cartilage tissue has poor self-regenerative ability, degeneration of cartilage in diseases like osteoarthritis presents limited options for repair and replacement. Thus, in this study, we focus on directing fhMSC to differentiate into chondrocytes for scalable cartilage tissue engineering. Here, we present a high throughput method of generating chondrogenic pellets in vitro with ultra-low attachment 96 well plates that are more spacesaving and cost effective for scale-up operations than conventional methods are. Our cytokine screen and dosage-dependent studies revealed that BMP2, when used at 50-100 ng/ml, is more effective in inducing the chondrogenic differentiation of fhMSC than TGFb1/3 are. These results are supported by functional output assays that measure glycosaminoglycan and type II collagen content, and gene expression analysis. Calcium quantification and histological staining further ascertained that BMP2 does not induce bone formation from fhMSC. In all, our results demonstrate that BMP2 induces effective chondrogenic differentiation of fhMSC, rather than bone differentiation. They also suggest that there are innate differences in hMSC derived from distinct sources to external signaling factors. Understanding how these differences are mediated would inform future work delving into scalable cartilage tissue engineering.
252 EVALUATION OF STEM CELL POPULATIONS ISOLATED FROM ADIPOSE TISSUE USING MINIMALLY MANIPULATED TECHNIQUES S Mehta3, F Silva2, V Vargas2, C Elabd2, C Crombie3, A Patel1 1 Cardiothoracic Surgery, University of Utah, Salt Lake City, Utah, United States, 2BioRestorative Therapies, Long Island, New York, United States, 3Plastic and Reconstructive Surgery, University of Utah, Salt Lake City, Utah, United States Rational: Autologous fat grafting is a highly used minimally manipulated technique in plastic and reconstructive surgery. Several fat-processing techniques have been described such as centrifugation, Telfa-rolling, blue-towel, sieve and decanting methods). Although studies have compared processing methods, no studies have evaluated the processing time, cost and stem cell potency of the different methods. Objective: Here we describe a study where we compare the processing time, cost, degree of cell viability and adipose derived stem cell (ADSC) potency of adipose tissue processed using several minimally manipulated fat-processing techniques adipose tissue. Methods and Results: Human adipose tissue was harvested and processed using centrifugation, Telfa-rolling, blue-towel, sieve and decanting methods. Processed tissue was placed into culture using ADSC cell culture methods to allow cells to grow. Cells were tested for doubling times, cell surface marker analysis and differentiation potential. Conclusions: All methods resulted in the isolation of adipose derived stem cells capable of undergoing adipo, osteo and chondrogenesis. Telfa-rolling and centrifuge methods resulted in the highest number of cells immediately after processing, and reached a cell density of 20 x106 within 6 days of tissue processing. Telfa-rolling is the most cost effective minimally manipulated method of processing adipose tissue that results in the highest ADSC isolation. This study supports larger clinical studies to evaluate the clinical benefits of using the Telfa-rolling method as the preferred minimally manipulated technique used in fat grafting.
253 ANALYSIS OF GENE EXPRESSIONS AND METABOLIC ACTIVITIES IN FETAL AND POSTNATAL HUMAN HEPATOCYTES FOR CLINICAL THERAPY R Gramignoli1, K Kannisto1, RC Srinivasan1, T Miki2, SC Strom1 1 Laboratory Medicine, Karolinska Institute, Stockholm, Sweden, 2Department of Biochemistry and Molecular Biology, University Southern California, Los Angeles, California, United States Fetal hepatocytes have been proposed for clinical hepatocyte transplants. However, to be useful, sufficient metabolic activities must be present. We assessed the viability and metabolic activities in isolated fetal human hepatocytes as compared to pediatric and adult hepatocytes, and we evaluated liver repopulation post-transplant in mice. Hepatocytes were isolated from 254 fetal livers, 11-24 weeks gestation and 229 post-natal tissues (3 months-85 years). Gene expression profile and functional tests (including Cytochrome P450 (CYP) activities, phase II-conjugation and ammonia metabolism) were assessed after isolation and long-term culture. Cells were transplanted to SCID/beige mice previously treated with retrorsine, and liver tissue analyzed after 1, 3 and 6 months for human DNA content (Alu sequences) and RNA levels of several liver genes. The viability of isolated fetal hepatocytes was significantly higher compared to adults (919% vs 7921%, p<0.0001). CYP1A, 2C9 and 3A4 activity increased with gestational age, but remained 1-6% of adult values. The fetal form of CYP3A (3A7) was expressed at high levels, and its activity decrease after birth. Phase II-conjugation and ammonia metabolism were null/low in fetal hepatocytes immediately after isolation, but increased to levels observed in mature hepatocytes in cells cultured for 5 days. DNA analysis and immunohistochemistry confirmed the presence of human hepatocytes in mouse liver. The average level of repopulation with fetal cells was 18-times higher than that observed with adult hepatocytes at 6 months post transplant. Once engrafted, the expression of several mature liver genes increased, however theynever exceeded 20% of normal adult levels. Fetal hepatocytes may be useful for clinical transplants; however they lack significant metabolic activities when isolated. Maturation of the cells may take longer than previously expected, and their activities need to be matched with patient requirements. 254 WILL NOT BE PRESENTED 255 CD271 ENRICHMENT OF BM-DERIVED MSCs ENHANCES THEIR OSTEOGENIC POTENCY S Müller, J Tang, L Nicholson, X Nong-Wang, JR De Havilland, A Dickinson Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom Mesenchymal stem cells (MSCs) are a promising candidate of regenerative medicine in treating bone destruction in osteoarthritis (OA). In vitro expansion of MSCs isolated via plastic adherence reduces their potential for osteogenic differentiation. CD271highCD45- MSCs, thought to be MSC progenitors, have been shown to be osteogenicly primed and may be better suited to bone regeneration. This study compared MSC enriched for CD271 (CD271-MSC) with non-enriched plastic adherent populations (PA-MSC) to address whether CD271-MSC have more potent bone forming capacity. From 5 samples of bone marrow derived mononuclear cells, samples pre and post CD271 enrichment were used to generate PA-MSCs and CD271-MSC respectively, which were subsequently cultured in parallel. At passages 2, 3 and 6 the samples were assessed for phenotypic profile by flow cytometry and differentiation potential following lineage induction and chemical staining. Osteogenic gene expression was measured using real-time PCR. CD271 enrichment resulted in an over 1000 fold increase in the frequency of CD271highCD45- cells, from 0.01% (0.003 SEM) in PA-MSCs to 11% (1.77 SEM) in CD271-MSCs. At all passages, each population expressed the standard MSC phenotype. CD271-MSCs had a slightly enhanced rate of population doubling, reaching 12 (0.13 SEM) population doublings by passage 6, while the PA-MSCs had undergone 11 (0.74 SEM) population doublings.