COMPARISON OF MOLECULAR AND FUNCTIONAL PROFILES OF WHARTON’S JELLY AND BONE MARROW MESENCHYMAL STEM CELLS
(Abstract release date: 05/19/16)
EHA Library. Pontikoglou C. 06/09/16; 132686; E1137
Prof. Charalampos Pontikoglou
Contributions
Contributions
Abstract
Abstract: E1137
Type: Eposter Presentation
Background
We have previously shown that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced differentiation potential to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. This altered differentiation capacity has been attributed, at least in part due to reduced expression of Wnt antagonist sFRP4 (a promoter of adipogenesis) and WNT-induced secreted protein-1 (a regulator of osteogenesis in WJ-MSCs), thereby supporting the contribution of WNT-pathways in human MSC biology (Batsali A et al.,Blood 2014:124,21).
Aims
In order to extent the comparative study between BM and WJ-MSCs we seek to estimate the telomere length of cultured MSCs and analyze the cell-cycle signaling pathway. Moreover, we aim to investigate the capacity of WJ-MSCs to support the growth of hematopoietic stem cells (HSCs).
Methods
MSCs were isolated and expanded from healthy donors’ BM aspirates (n=15) and from the WJ of full-term neonates (n=15) after written informed consent. MSCs were in vitro expanded and re-seeded for a total of 10 passages and were verified phenotypically by flow cytometry. The expression of 84 genes related to cell-cycle signaling pathway was assessed using a PCR array (Fold Change (FC) =2-ΔΔCt). DNA was isolated from culture expanded MSCs (at P2, P6 and P10) and telomere length was measured by means of real-time-PCR using β-globin as control. Telomere length is proportional to the relative telomere/single-copy-gene ratio, T/S= 2-ΔCt (ΔCt= Cttelomere - Ctβ-globin). Levels of human hematopoietic associated cytokines (Flt-3 ligand/G-CSF/GM-CSF/SDF-1a) were measured by means of enzyme-linked-immunosorbent assay, from supernatants of MSC cultures (from P2, P6 and P10). MSC hematopoietic supportive capacity was evaluated in two-week co-cultures with allogeneic BM or UC-blood CD34+ cells.
Results
Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. Regarding the cell-cycle signaling pathway many genes essential for mitosis and genome stability were up-regulated in WJ-MSCs, such as ATM (FC=4.04), CCND2 (FC=4.55), AURKB (FC=3.15) and MKI67 (FC=2.86), while the anti-apoptotic gene BCL2 was down-regulated (FC=4.07). Moreover the relative telomere length of WJ-MSCs was higher comparing with BM-MSCs through all passages, with a significant difference in P2 (p=0.032). The expression of hematopoietic associated cytokine Flt-3 ligand, an important cytokine for development of several hematopoietic populations, was expressed in both MSC-sources. G-CSF and GM-CSF, growth factors of HSCs involved in the production of granulocytes, macrophages and dendritic cells, were significantly up-regulated in WJ-supernatants but were almost absent in BM-cultures (p<0.001). The regulator of migration, adherence and engraftment of HSCs, SDF-1a, was significantly overexpressed in BM-supernatants through cultivation (p<0.05). Nevertheless, WJ-MSCs seemed not to support CD34+ cell growth as evidenced by the numbers of total colony-forming units (CFUs), at least in the current experimental setting.
Conclusion
Collectively, our results suggest that WJ-MSC population exhibit greater proliferation potential and decreased apoptosis, longer telomeric length and higher genome stability. The observed differences of CFUs in the co-culture systems could be attributed to the differences in cytokines secreted by WJ and BM-MSCs. Our results point out that WJ- and BM-MSCs exhibit unique characteristics and therefore the appropriate source should be selected for a specific clinical application.
Session topic: E-poster
Keyword(s): Hematopoiesis, Mesenchymal stem cell, Telomere length
Type: Eposter Presentation
Background
We have previously shown that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced differentiation potential to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. This altered differentiation capacity has been attributed, at least in part due to reduced expression of Wnt antagonist sFRP4 (a promoter of adipogenesis) and WNT-induced secreted protein-1 (a regulator of osteogenesis in WJ-MSCs), thereby supporting the contribution of WNT-pathways in human MSC biology (Batsali A et al.,Blood 2014:124,21).
Aims
In order to extent the comparative study between BM and WJ-MSCs we seek to estimate the telomere length of cultured MSCs and analyze the cell-cycle signaling pathway. Moreover, we aim to investigate the capacity of WJ-MSCs to support the growth of hematopoietic stem cells (HSCs).
Methods
MSCs were isolated and expanded from healthy donors’ BM aspirates (n=15) and from the WJ of full-term neonates (n=15) after written informed consent. MSCs were in vitro expanded and re-seeded for a total of 10 passages and were verified phenotypically by flow cytometry. The expression of 84 genes related to cell-cycle signaling pathway was assessed using a PCR array (Fold Change (FC) =2-ΔΔCt). DNA was isolated from culture expanded MSCs (at P2, P6 and P10) and telomere length was measured by means of real-time-PCR using β-globin as control. Telomere length is proportional to the relative telomere/single-copy-gene ratio, T/S= 2-ΔCt (ΔCt= Cttelomere - Ctβ-globin). Levels of human hematopoietic associated cytokines (Flt-3 ligand/G-CSF/GM-CSF/SDF-1a) were measured by means of enzyme-linked-immunosorbent assay, from supernatants of MSC cultures (from P2, P6 and P10). MSC hematopoietic supportive capacity was evaluated in two-week co-cultures with allogeneic BM or UC-blood CD34+ cells.
Results
Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. Regarding the cell-cycle signaling pathway many genes essential for mitosis and genome stability were up-regulated in WJ-MSCs, such as ATM (FC=4.04), CCND2 (FC=4.55), AURKB (FC=3.15) and MKI67 (FC=2.86), while the anti-apoptotic gene BCL2 was down-regulated (FC=4.07). Moreover the relative telomere length of WJ-MSCs was higher comparing with BM-MSCs through all passages, with a significant difference in P2 (p=0.032). The expression of hematopoietic associated cytokine Flt-3 ligand, an important cytokine for development of several hematopoietic populations, was expressed in both MSC-sources. G-CSF and GM-CSF, growth factors of HSCs involved in the production of granulocytes, macrophages and dendritic cells, were significantly up-regulated in WJ-supernatants but were almost absent in BM-cultures (p<0.001). The regulator of migration, adherence and engraftment of HSCs, SDF-1a, was significantly overexpressed in BM-supernatants through cultivation (p<0.05). Nevertheless, WJ-MSCs seemed not to support CD34+ cell growth as evidenced by the numbers of total colony-forming units (CFUs), at least in the current experimental setting.
Conclusion
Collectively, our results suggest that WJ-MSC population exhibit greater proliferation potential and decreased apoptosis, longer telomeric length and higher genome stability. The observed differences of CFUs in the co-culture systems could be attributed to the differences in cytokines secreted by WJ and BM-MSCs. Our results point out that WJ- and BM-MSCs exhibit unique characteristics and therefore the appropriate source should be selected for a specific clinical application.
Session topic: E-poster
Keyword(s): Hematopoiesis, Mesenchymal stem cell, Telomere length
Abstract: E1137
Type: Eposter Presentation
Background
We have previously shown that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced differentiation potential to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. This altered differentiation capacity has been attributed, at least in part due to reduced expression of Wnt antagonist sFRP4 (a promoter of adipogenesis) and WNT-induced secreted protein-1 (a regulator of osteogenesis in WJ-MSCs), thereby supporting the contribution of WNT-pathways in human MSC biology (Batsali A et al.,Blood 2014:124,21).
Aims
In order to extent the comparative study between BM and WJ-MSCs we seek to estimate the telomere length of cultured MSCs and analyze the cell-cycle signaling pathway. Moreover, we aim to investigate the capacity of WJ-MSCs to support the growth of hematopoietic stem cells (HSCs).
Methods
MSCs were isolated and expanded from healthy donors’ BM aspirates (n=15) and from the WJ of full-term neonates (n=15) after written informed consent. MSCs were in vitro expanded and re-seeded for a total of 10 passages and were verified phenotypically by flow cytometry. The expression of 84 genes related to cell-cycle signaling pathway was assessed using a PCR array (Fold Change (FC) =2-ΔΔCt). DNA was isolated from culture expanded MSCs (at P2, P6 and P10) and telomere length was measured by means of real-time-PCR using β-globin as control. Telomere length is proportional to the relative telomere/single-copy-gene ratio, T/S= 2-ΔCt (ΔCt= Cttelomere - Ctβ-globin). Levels of human hematopoietic associated cytokines (Flt-3 ligand/G-CSF/GM-CSF/SDF-1a) were measured by means of enzyme-linked-immunosorbent assay, from supernatants of MSC cultures (from P2, P6 and P10). MSC hematopoietic supportive capacity was evaluated in two-week co-cultures with allogeneic BM or UC-blood CD34+ cells.
Results
Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. Regarding the cell-cycle signaling pathway many genes essential for mitosis and genome stability were up-regulated in WJ-MSCs, such as ATM (FC=4.04), CCND2 (FC=4.55), AURKB (FC=3.15) and MKI67 (FC=2.86), while the anti-apoptotic gene BCL2 was down-regulated (FC=4.07). Moreover the relative telomere length of WJ-MSCs was higher comparing with BM-MSCs through all passages, with a significant difference in P2 (p=0.032). The expression of hematopoietic associated cytokine Flt-3 ligand, an important cytokine for development of several hematopoietic populations, was expressed in both MSC-sources. G-CSF and GM-CSF, growth factors of HSCs involved in the production of granulocytes, macrophages and dendritic cells, were significantly up-regulated in WJ-supernatants but were almost absent in BM-cultures (p<0.001). The regulator of migration, adherence and engraftment of HSCs, SDF-1a, was significantly overexpressed in BM-supernatants through cultivation (p<0.05). Nevertheless, WJ-MSCs seemed not to support CD34+ cell growth as evidenced by the numbers of total colony-forming units (CFUs), at least in the current experimental setting.
Conclusion
Collectively, our results suggest that WJ-MSC population exhibit greater proliferation potential and decreased apoptosis, longer telomeric length and higher genome stability. The observed differences of CFUs in the co-culture systems could be attributed to the differences in cytokines secreted by WJ and BM-MSCs. Our results point out that WJ- and BM-MSCs exhibit unique characteristics and therefore the appropriate source should be selected for a specific clinical application.
Session topic: E-poster
Keyword(s): Hematopoiesis, Mesenchymal stem cell, Telomere length
Type: Eposter Presentation
Background
We have previously shown that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced differentiation potential to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. This altered differentiation capacity has been attributed, at least in part due to reduced expression of Wnt antagonist sFRP4 (a promoter of adipogenesis) and WNT-induced secreted protein-1 (a regulator of osteogenesis in WJ-MSCs), thereby supporting the contribution of WNT-pathways in human MSC biology (Batsali A et al.,Blood 2014:124,21).
Aims
In order to extent the comparative study between BM and WJ-MSCs we seek to estimate the telomere length of cultured MSCs and analyze the cell-cycle signaling pathway. Moreover, we aim to investigate the capacity of WJ-MSCs to support the growth of hematopoietic stem cells (HSCs).
Methods
MSCs were isolated and expanded from healthy donors’ BM aspirates (n=15) and from the WJ of full-term neonates (n=15) after written informed consent. MSCs were in vitro expanded and re-seeded for a total of 10 passages and were verified phenotypically by flow cytometry. The expression of 84 genes related to cell-cycle signaling pathway was assessed using a PCR array (Fold Change (FC) =2-ΔΔCt). DNA was isolated from culture expanded MSCs (at P2, P6 and P10) and telomere length was measured by means of real-time-PCR using β-globin as control. Telomere length is proportional to the relative telomere/single-copy-gene ratio, T/S= 2-ΔCt (ΔCt= Cttelomere - Ctβ-globin). Levels of human hematopoietic associated cytokines (Flt-3 ligand/G-CSF/GM-CSF/SDF-1a) were measured by means of enzyme-linked-immunosorbent assay, from supernatants of MSC cultures (from P2, P6 and P10). MSC hematopoietic supportive capacity was evaluated in two-week co-cultures with allogeneic BM or UC-blood CD34+ cells.
Results
Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. Regarding the cell-cycle signaling pathway many genes essential for mitosis and genome stability were up-regulated in WJ-MSCs, such as ATM (FC=4.04), CCND2 (FC=4.55), AURKB (FC=3.15) and MKI67 (FC=2.86), while the anti-apoptotic gene BCL2 was down-regulated (FC=4.07). Moreover the relative telomere length of WJ-MSCs was higher comparing with BM-MSCs through all passages, with a significant difference in P2 (p=0.032). The expression of hematopoietic associated cytokine Flt-3 ligand, an important cytokine for development of several hematopoietic populations, was expressed in both MSC-sources. G-CSF and GM-CSF, growth factors of HSCs involved in the production of granulocytes, macrophages and dendritic cells, were significantly up-regulated in WJ-supernatants but were almost absent in BM-cultures (p<0.001). The regulator of migration, adherence and engraftment of HSCs, SDF-1a, was significantly overexpressed in BM-supernatants through cultivation (p<0.05). Nevertheless, WJ-MSCs seemed not to support CD34+ cell growth as evidenced by the numbers of total colony-forming units (CFUs), at least in the current experimental setting.
Conclusion
Collectively, our results suggest that WJ-MSC population exhibit greater proliferation potential and decreased apoptosis, longer telomeric length and higher genome stability. The observed differences of CFUs in the co-culture systems could be attributed to the differences in cytokines secreted by WJ and BM-MSCs. Our results point out that WJ- and BM-MSCs exhibit unique characteristics and therefore the appropriate source should be selected for a specific clinical application.
Session topic: E-poster
Keyword(s): Hematopoiesis, Mesenchymal stem cell, Telomere length
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