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Medium supplementation with BMP-4 increases activin a gene expression during osteogenic differentiation of mouse embryonic stem cells
stem cell markers and proved for multilineage differentiation potential. The gene expression was analyzed by RT-PCR. RESULTS: The fresh hGCs showed short and flat cell morphology. The hGSCs not only characterized with long-term proliferating ability, they also expressed vimentin, embryonic stem cell markers (Oct-4 and Nanog) and mesenchymal stem cell markers (CD44, CD90 and CD105). In cell proliferative potential, hGSCs could be maintained for 3 months and presented the higher cumulative population doublings compared with hGCs. For the cell differentiation, the hGSCs were capable of adipogenesis by Oil Red O staining, osteogenesis by von Kossa staining, and chondrogenesis by Alcian blue staining. After FSH treatment for 21 days, the hGSCs highly expressed StAR and CYP11A genes which regulate the initial step of steroidogenesis from cholesterol to pregnenolone. CONCLUSION: In this study, we demonstrated that hGSCs can be isolated and maintained in vitro. The steroidogenic genes can be induced by FSH treatment. Therefore, hGSCs may be a good source for stem cell therapy and this culture model also served as a platform for future drug discovery.
P-1065 Thursday, October 17, 2013 SAPK ACTIVITY IS A BETTER MARKER THAN AMPK ACTIVITY FOR MEASURING STRESSFUL O2 LEVELS IN STEM CELLS. E. E. Puscheck,a Z. Jiang,a Y. F. Xie,a S. C. Zhou,b D. A. Rappolee.a,b aOB/GYN, Wayne State University School of Medicine, Detroit, MI; bReproductive Sciences/physiology, Wayne Stete University School of Medicine, Detroit, MI. OBJECTIVE: To test whether stress-activated protein kinase (SAPK) or AMP-activated protein kinase (AMPK) correlates with stress as induced by non-physiologic O2 levels during culture of mouse trophoblast stem cells (TSC). DESIGN: Experimental. MATERIALS AND METHODS: TSC were cultured at four O2 concentrations (20, 2, 0.5, or 0%), with fibroblast growth factor (FGF)-4 present to maintain stemness. We measured stress responses by 3 mechanisms: 1. Quantified the magnitude of AMPK and SAPK enzyme activity levels, 2. Measured kinetics by rates of stress enzyme activation after O2 switches (from 2-20% or 2-0% and 20-2% or 20-0%), 3. Quantified growth by measuring stem cell accumulation. Immunoblots were used to analyze O2dependent changes in magnitude and rate of change in stress enzyme activity. RESULTS: Decreasing O2 (from 20 to 0%) increased AMPK activity in an S-shaped response as prior reports of other stressors showed at 20% O2. Unlike prior reports, SAPK activity produced a U-shaped response in the 20-0% O2, which allows us to identify a specific minimum stress to TSC cultured at 2% O2. The U-shaped response for SAPK activity was inversely proportional growth of stem cells, resulting in an inverted U-shaped response for stem cell accumulation. The fastest change in SAPK and AMPK activity was during 220% or 2-0% O2 switches, suggesting that 2% O2 was optimal and deviation from this required the fastest response. CONCLUSION: SAPK activity (stress) was inversely proportional to growth at improper O2 levels as reported previously for two stressors during TSC culture at 20% O2, but here AMPK was inversely proportional to growth only at O2 levels <2%. Rate of change corresponded to departure from the stress minimum for SAPK activity, corroborating the stress minimum at 2% O2. Thus, SAPK more accurately reports the O2 optimum for TSC and should be the best stress reporter for blastocysts. Supported by: R03HD061431.
DESIGN: Isolation and cultivation of mouse ICM-derived stem cells. MATERIALS AND METHODS: Stem cells were isolated by laser biopsy of blastocysts or alternatively by plating zona-free whole blastocysts, followed by selective passaging. DMEM with 20% fetal calf serum, LIF (10 ng/ml), glutamine and non-essential amino acids was used for cutlture. Human endometrial cells (HEC) were seeded in to a multi-well plate with Transwell membrane inserts. ICMs or whole blastocysts were plated on the membrane inserts. Cultures were passaged 2-3 times /week. ICM derived stem colonies were tested for expression of stem cell specific markers alkaline phosphatase (AP), SSEA-1, Oct-4 and SOX-2. RESULTS: Both isolation techniques allowed derivation and continued propagation of stem cells. Colonies exhibited typical embryonic stem cell (ESC) morphology. To date the ICM-derived stem cells have been propagated for 17 passages and 9 weeks in culture and continue to express stem cell markers SSEA-1, Sox-2, Oct-4 and alkaline phosphatase. Passaged stem cells also retained their ESC characteristics after freezing and thawing. Our data suggest that the HEC feeder is capable of sustaining the pluripotency of embryonic stem cells derived from the ICMs. Timely passaging was critical as colonies with > 150 mm diameter before passaging showed evidence of pre-differentiation around the borders .Separation of the stem cells from the human feeder layer simplified ESC passaging. CONCLUSION: This non-contact co-culture model with a human endometrial feeder layer may be useful for deriving stem cell lines from blastocysts. Application of this system to human stem cell derivation is needed as further proof of concept.
P-1067 Thursday, October 17, 2013 MEDIUM SUPPLEMENTATION WITH BMP-4 INCREASES ACTIVIN A GENE EXPRESSION DURING OSTEOGENIC DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS. R. L. Tavares, B. M. Camargos, H. L. Del Puerto, A. F. Camargos, F. M. Reis. Obstetrics and Gynecology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. OBJECTIVE: To study the BMP-4 medium supplemetation effect on Activin A gene expression during in vitro osteogenic diferentiation of mouse embryonic stem cells (ESC). DESIGN: Prospective study. MATERIALS AND METHODS: A newly isololated mouse ESC which was differentiated into osteogenic precursors. Real time PCR was performed for RUNX2, Bone Alkaline Phosphatase, osteocalcin and activin A receptors. We correlated the expression of Activin A in osteogenic precursors with and without BMP-4 culture. RESULTS: Gene expression of activin A is expressed in undifferentiated mouse ESC, and gradually increases with time of cell culture, particularly with the use of BMP-4, during in vitro ESC osteogenec differentiation. The osteogenic differentiation protocol used revealed some gene expression of undifferentiation markers even in the final stage of differentiation, which can be a limiting factor for use in cell therapy. CONCLUSION: Activin A may play a role on the undifferentiation state of mouse embryonic stem cells in vitro. Activin A may be important during in vitro osteogenesis of mouse ES cells, and its activity is increased by BMP-4 during cell differentiation into bone precursors. Supported by: The National Intitutes of Hormones and Women’s Health. Federal University of Minas Gerais.
P-1068 Thursday, October 17, 2013 P-1066 Thursday, October 17, 2013 TECHNIQUE FOR DERIVATION OF STEM CELL LINES FROM THE INNER CELL MASS OF MOUSE BLASTOCSYTS USING A NOVEL HUMAN ENDOMETRIAL CELL LINE AND A NON-CONTACT CO-CULTURE SYSTEM. N. Desai, J. Ludgin. OB-GYN/Women’s Health Institute, Cleveland Clinic, Beachwood, OH. OBJECTIVE: Therapeutic use of stem cells may require the derivation and propagation of clinical grade stem cell lines using non-animal feeder layers. To this end, we test a novel human endometrial cell (HEC) line as a feeder layer and its ability to support the growth of stem cells isolated from the inner cell mass (ICM) of mouse blastocsyts.
S456
ASRM Abstracts
COMPARATIVE ANALYSIS OF THE LIPID PROFILE OF HUMAN MESENCHYMAL STEM CELLS INDUCED TO PLURIPOTENCY BY DIFFERENT TRANSFECTION FACTORS. R. Rochetti,a F. F. Bressan,b T. Regiani,c C. A. Labate,c R. Fraietta,a F. V. Meirelles.b a Department of Surgery, Division of Urology, Human Reproduction Section, S~ao Paulo Federal University, S~ao Paulo, Brazil; bDepartment of Basic Science, University of S~ao Paulo, Pirassununga, S~ao Paulo, Brazil; cDepartment of Genetics, University of S~ao Paulo, Piracicaba, S~ao Paulo, Brazil. OBJECTIVE: Studies of induced pluripotent stem (iPS) cells have growing to answer questions about the proceedings arising of reprogramming. This process is also inefficient becoming a handicap for mechanistic
Vol. 100, No. 3, Supplement, September 2013
P-1065 Thursday, October 17, 2013 SAPK ACTIVITY IS A BETTER MARKER THAN AMPK ACTIVITY FOR MEASURING STRESSFUL O2 LEVELS IN STEM CELLS. E. E. Puscheck,a Z. Jiang,a Y. F. Xie,a S. C. Zhou,b D. A. Rappolee.a,b aOB/GYN, Wayne State University School of Medicine, Detroit, MI; bReproductive Sciences/physiology, Wayne Stete University School of Medicine, Detroit, MI. OBJECTIVE: To test whether stress-activated protein kinase (SAPK) or AMP-activated protein kinase (AMPK) correlates with stress as induced by non-physiologic O2 levels during culture of mouse trophoblast stem cells (TSC). DESIGN: Experimental. MATERIALS AND METHODS: TSC were cultured at four O2 concentrations (20, 2, 0.5, or 0%), with fibroblast growth factor (FGF)-4 present to maintain stemness. We measured stress responses by 3 mechanisms: 1. Quantified the magnitude of AMPK and SAPK enzyme activity levels, 2. Measured kinetics by rates of stress enzyme activation after O2 switches (from 2-20% or 2-0% and 20-2% or 20-0%), 3. Quantified growth by measuring stem cell accumulation. Immunoblots were used to analyze O2dependent changes in magnitude and rate of change in stress enzyme activity. RESULTS: Decreasing O2 (from 20 to 0%) increased AMPK activity in an S-shaped response as prior reports of other stressors showed at 20% O2. Unlike prior reports, SAPK activity produced a U-shaped response in the 20-0% O2, which allows us to identify a specific minimum stress to TSC cultured at 2% O2. The U-shaped response for SAPK activity was inversely proportional growth of stem cells, resulting in an inverted U-shaped response for stem cell accumulation. The fastest change in SAPK and AMPK activity was during 220% or 2-0% O2 switches, suggesting that 2% O2 was optimal and deviation from this required the fastest response. CONCLUSION: SAPK activity (stress) was inversely proportional to growth at improper O2 levels as reported previously for two stressors during TSC culture at 20% O2, but here AMPK was inversely proportional to growth only at O2 levels <2%. Rate of change corresponded to departure from the stress minimum for SAPK activity, corroborating the stress minimum at 2% O2. Thus, SAPK more accurately reports the O2 optimum for TSC and should be the best stress reporter for blastocysts. Supported by: R03HD061431.
DESIGN: Isolation and cultivation of mouse ICM-derived stem cells. MATERIALS AND METHODS: Stem cells were isolated by laser biopsy of blastocysts or alternatively by plating zona-free whole blastocysts, followed by selective passaging. DMEM with 20% fetal calf serum, LIF (10 ng/ml), glutamine and non-essential amino acids was used for cutlture. Human endometrial cells (HEC) were seeded in to a multi-well plate with Transwell membrane inserts. ICMs or whole blastocysts were plated on the membrane inserts. Cultures were passaged 2-3 times /week. ICM derived stem colonies were tested for expression of stem cell specific markers alkaline phosphatase (AP), SSEA-1, Oct-4 and SOX-2. RESULTS: Both isolation techniques allowed derivation and continued propagation of stem cells. Colonies exhibited typical embryonic stem cell (ESC) morphology. To date the ICM-derived stem cells have been propagated for 17 passages and 9 weeks in culture and continue to express stem cell markers SSEA-1, Sox-2, Oct-4 and alkaline phosphatase. Passaged stem cells also retained their ESC characteristics after freezing and thawing. Our data suggest that the HEC feeder is capable of sustaining the pluripotency of embryonic stem cells derived from the ICMs. Timely passaging was critical as colonies with > 150 mm diameter before passaging showed evidence of pre-differentiation around the borders .Separation of the stem cells from the human feeder layer simplified ESC passaging. CONCLUSION: This non-contact co-culture model with a human endometrial feeder layer may be useful for deriving stem cell lines from blastocysts. Application of this system to human stem cell derivation is needed as further proof of concept.
P-1067 Thursday, October 17, 2013 MEDIUM SUPPLEMENTATION WITH BMP-4 INCREASES ACTIVIN A GENE EXPRESSION DURING OSTEOGENIC DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS. R. L. Tavares, B. M. Camargos, H. L. Del Puerto, A. F. Camargos, F. M. Reis. Obstetrics and Gynecology, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. OBJECTIVE: To study the BMP-4 medium supplemetation effect on Activin A gene expression during in vitro osteogenic diferentiation of mouse embryonic stem cells (ESC). DESIGN: Prospective study. MATERIALS AND METHODS: A newly isololated mouse ESC which was differentiated into osteogenic precursors. Real time PCR was performed for RUNX2, Bone Alkaline Phosphatase, osteocalcin and activin A receptors. We correlated the expression of Activin A in osteogenic precursors with and without BMP-4 culture. RESULTS: Gene expression of activin A is expressed in undifferentiated mouse ESC, and gradually increases with time of cell culture, particularly with the use of BMP-4, during in vitro ESC osteogenec differentiation. The osteogenic differentiation protocol used revealed some gene expression of undifferentiation markers even in the final stage of differentiation, which can be a limiting factor for use in cell therapy. CONCLUSION: Activin A may play a role on the undifferentiation state of mouse embryonic stem cells in vitro. Activin A may be important during in vitro osteogenesis of mouse ES cells, and its activity is increased by BMP-4 during cell differentiation into bone precursors. Supported by: The National Intitutes of Hormones and Women’s Health. Federal University of Minas Gerais.
P-1068 Thursday, October 17, 2013 P-1066 Thursday, October 17, 2013 TECHNIQUE FOR DERIVATION OF STEM CELL LINES FROM THE INNER CELL MASS OF MOUSE BLASTOCSYTS USING A NOVEL HUMAN ENDOMETRIAL CELL LINE AND A NON-CONTACT CO-CULTURE SYSTEM. N. Desai, J. Ludgin. OB-GYN/Women’s Health Institute, Cleveland Clinic, Beachwood, OH. OBJECTIVE: Therapeutic use of stem cells may require the derivation and propagation of clinical grade stem cell lines using non-animal feeder layers. To this end, we test a novel human endometrial cell (HEC) line as a feeder layer and its ability to support the growth of stem cells isolated from the inner cell mass (ICM) of mouse blastocsyts.
S456
ASRM Abstracts
COMPARATIVE ANALYSIS OF THE LIPID PROFILE OF HUMAN MESENCHYMAL STEM CELLS INDUCED TO PLURIPOTENCY BY DIFFERENT TRANSFECTION FACTORS. R. Rochetti,a F. F. Bressan,b T. Regiani,c C. A. Labate,c R. Fraietta,a F. V. Meirelles.b a Department of Surgery, Division of Urology, Human Reproduction Section, S~ao Paulo Federal University, S~ao Paulo, Brazil; bDepartment of Basic Science, University of S~ao Paulo, Pirassununga, S~ao Paulo, Brazil; cDepartment of Genetics, University of S~ao Paulo, Piracicaba, S~ao Paulo, Brazil. OBJECTIVE: Studies of induced pluripotent stem (iPS) cells have growing to answer questions about the proceedings arising of reprogramming. This process is also inefficient becoming a handicap for mechanistic
Vol. 100, No. 3, Supplement, September 2013