BONE MARROW MESENCHYMAL STEM/STROMAL CELLS FROM SPLENIC MARGINAL ZONE LYMPHOMA PATIENTS SHOW DISTINCT INTRINSIC CHARACTERISTICS AND EFFECTS ON B-LYMPHOCYTE CHEMOTAXIS AND APOPTOSIS
(Abstract release date: 05/19/16)
EHA Library. Pontikoglou C. 06/09/16; 132927; E1378
Prof. Charalampos Pontikoglou
Contributions
Contributions
Abstract
Abstract: E1378
Type: Eposter Presentation
Background
Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. In most cases, B-lymphocytes infiltrate bone marrow (BM), suggesting a potential involvement of BM microenvironment in disease pathology.
Aims
To examine BM-derived mesenchymal stem/stromal cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if they exhibit altered intrinsic properties in vitro or if they stimulate altered B- and T-cell immunomodulatory properties in SMZL patients compared to healthy controls.
Methods
BM MSCs were isolated and expanded in vitro from 12 SMZL patients and 11 healthy controls. MSCs were cultured for a total of 5 passages (P) and were phenotypically characterized by flow cytometry (FC). The colony forming unit-fibroblast (CFU-F) assay was used to estimate MSC frequency within the BM mononuclear cell (BMMC) fraction in P0 and their clonogenic capacity in P2. MSCs differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. Their proliferative potential was evaluated by Methyl Triazolyl Tetrazolium assay. To assess the effect of SMZL MSCs on lymphocyte proliferation, B- and T-cells were immunomagnetically isolated (Miltenyi Biotec) from peripheral blood (PB) of normal individuals, labeled with carboxyfluorescein succinimidyl ester (Gibco) and subsequently cultured in the absence or presence of confluent layers of SMZL or normal MSCs, in the presence of activating factors. B cell apoptosis was evaluated via FC using 7-AAD staining, after co-culturing with SMZL or normal MSCs. Finally, B-cell chemotaxis was evaluated by monitoring the migration of isolated B-cells through a 5µm pore membrane in a transwell plate towards the confluent layers of either SMZL or normal MSCs.
Results
In vitro expanded MSCs from both groups were adherent cells with a spindle-shape morphology, which expressed CD29, CD73, CD90 and CD105 and were negative for the CD14, CD34 and CD45 cell surface markers. SMZL and Normal MSCs were similar in their differentiation to adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.51 vs. 7.95 colonies per 105 ΒΜΜCs respectively; p=0.04) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs in comparison to Normal MSCs at P2, displayed defective clonogenic potential (3.36 vs. 6.23 colonies respectively, p=0.03) and reduced proliferative potential (p<0.03).In co-culture experiments, SMZL and Normal MSCs showed no difference in their effect on normal B-cell or T-cell proliferation. However, SMZL MSCs produced a statistically significant advantage on B-cell survival as measured in B-cell apoptosis experiments. More specifically, 50±1.46% of B cells cultured in medium alone were apoptotic, while only 23±2.63% and 17±0.77% of B cells co-cultured with either Normal MSCs or SMZL MSCs were apoptotic (p=0.009 when comparing SMZL vs Normal MSCs). Finally the transwell migration assays showed that SMZL MSCs displayed a stronger chemotactic activity on isolated normal B-cells, in comparison to Normal MSCs, with the percentages of migrated B-cells being 34% and 22% respectively, (p=0.009).
Conclusion
Our data suggest that SMZL MSCs are intrinsically defective in terms of proliferative and clonogenic potential and moreover they exert distint anti-apoptotic and chemotactic effects on healthy-donor derived B cells. The impact of SMZL MSCs on the properties of patient derived peripheral B cells is currently under investigation.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell, Microenvironment, Splenic marginal zone lymphoma
Type: Eposter Presentation
Background
Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. In most cases, B-lymphocytes infiltrate bone marrow (BM), suggesting a potential involvement of BM microenvironment in disease pathology.
Aims
To examine BM-derived mesenchymal stem/stromal cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if they exhibit altered intrinsic properties in vitro or if they stimulate altered B- and T-cell immunomodulatory properties in SMZL patients compared to healthy controls.
Methods
BM MSCs were isolated and expanded in vitro from 12 SMZL patients and 11 healthy controls. MSCs were cultured for a total of 5 passages (P) and were phenotypically characterized by flow cytometry (FC). The colony forming unit-fibroblast (CFU-F) assay was used to estimate MSC frequency within the BM mononuclear cell (BMMC) fraction in P0 and their clonogenic capacity in P2. MSCs differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. Their proliferative potential was evaluated by Methyl Triazolyl Tetrazolium assay. To assess the effect of SMZL MSCs on lymphocyte proliferation, B- and T-cells were immunomagnetically isolated (Miltenyi Biotec) from peripheral blood (PB) of normal individuals, labeled with carboxyfluorescein succinimidyl ester (Gibco) and subsequently cultured in the absence or presence of confluent layers of SMZL or normal MSCs, in the presence of activating factors. B cell apoptosis was evaluated via FC using 7-AAD staining, after co-culturing with SMZL or normal MSCs. Finally, B-cell chemotaxis was evaluated by monitoring the migration of isolated B-cells through a 5µm pore membrane in a transwell plate towards the confluent layers of either SMZL or normal MSCs.
Results
In vitro expanded MSCs from both groups were adherent cells with a spindle-shape morphology, which expressed CD29, CD73, CD90 and CD105 and were negative for the CD14, CD34 and CD45 cell surface markers. SMZL and Normal MSCs were similar in their differentiation to adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.51 vs. 7.95 colonies per 105 ΒΜΜCs respectively; p=0.04) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs in comparison to Normal MSCs at P2, displayed defective clonogenic potential (3.36 vs. 6.23 colonies respectively, p=0.03) and reduced proliferative potential (p<0.03).In co-culture experiments, SMZL and Normal MSCs showed no difference in their effect on normal B-cell or T-cell proliferation. However, SMZL MSCs produced a statistically significant advantage on B-cell survival as measured in B-cell apoptosis experiments. More specifically, 50±1.46% of B cells cultured in medium alone were apoptotic, while only 23±2.63% and 17±0.77% of B cells co-cultured with either Normal MSCs or SMZL MSCs were apoptotic (p=0.009 when comparing SMZL vs Normal MSCs). Finally the transwell migration assays showed that SMZL MSCs displayed a stronger chemotactic activity on isolated normal B-cells, in comparison to Normal MSCs, with the percentages of migrated B-cells being 34% and 22% respectively, (p=0.009).
Conclusion
Our data suggest that SMZL MSCs are intrinsically defective in terms of proliferative and clonogenic potential and moreover they exert distint anti-apoptotic and chemotactic effects on healthy-donor derived B cells. The impact of SMZL MSCs on the properties of patient derived peripheral B cells is currently under investigation.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell, Microenvironment, Splenic marginal zone lymphoma
Abstract: E1378
Type: Eposter Presentation
Background
Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. In most cases, B-lymphocytes infiltrate bone marrow (BM), suggesting a potential involvement of BM microenvironment in disease pathology.
Aims
To examine BM-derived mesenchymal stem/stromal cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if they exhibit altered intrinsic properties in vitro or if they stimulate altered B- and T-cell immunomodulatory properties in SMZL patients compared to healthy controls.
Methods
BM MSCs were isolated and expanded in vitro from 12 SMZL patients and 11 healthy controls. MSCs were cultured for a total of 5 passages (P) and were phenotypically characterized by flow cytometry (FC). The colony forming unit-fibroblast (CFU-F) assay was used to estimate MSC frequency within the BM mononuclear cell (BMMC) fraction in P0 and their clonogenic capacity in P2. MSCs differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. Their proliferative potential was evaluated by Methyl Triazolyl Tetrazolium assay. To assess the effect of SMZL MSCs on lymphocyte proliferation, B- and T-cells were immunomagnetically isolated (Miltenyi Biotec) from peripheral blood (PB) of normal individuals, labeled with carboxyfluorescein succinimidyl ester (Gibco) and subsequently cultured in the absence or presence of confluent layers of SMZL or normal MSCs, in the presence of activating factors. B cell apoptosis was evaluated via FC using 7-AAD staining, after co-culturing with SMZL or normal MSCs. Finally, B-cell chemotaxis was evaluated by monitoring the migration of isolated B-cells through a 5µm pore membrane in a transwell plate towards the confluent layers of either SMZL or normal MSCs.
Results
In vitro expanded MSCs from both groups were adherent cells with a spindle-shape morphology, which expressed CD29, CD73, CD90 and CD105 and were negative for the CD14, CD34 and CD45 cell surface markers. SMZL and Normal MSCs were similar in their differentiation to adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.51 vs. 7.95 colonies per 105 ΒΜΜCs respectively; p=0.04) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs in comparison to Normal MSCs at P2, displayed defective clonogenic potential (3.36 vs. 6.23 colonies respectively, p=0.03) and reduced proliferative potential (p<0.03).In co-culture experiments, SMZL and Normal MSCs showed no difference in their effect on normal B-cell or T-cell proliferation. However, SMZL MSCs produced a statistically significant advantage on B-cell survival as measured in B-cell apoptosis experiments. More specifically, 50±1.46% of B cells cultured in medium alone were apoptotic, while only 23±2.63% and 17±0.77% of B cells co-cultured with either Normal MSCs or SMZL MSCs were apoptotic (p=0.009 when comparing SMZL vs Normal MSCs). Finally the transwell migration assays showed that SMZL MSCs displayed a stronger chemotactic activity on isolated normal B-cells, in comparison to Normal MSCs, with the percentages of migrated B-cells being 34% and 22% respectively, (p=0.009).
Conclusion
Our data suggest that SMZL MSCs are intrinsically defective in terms of proliferative and clonogenic potential and moreover they exert distint anti-apoptotic and chemotactic effects on healthy-donor derived B cells. The impact of SMZL MSCs on the properties of patient derived peripheral B cells is currently under investigation.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell, Microenvironment, Splenic marginal zone lymphoma
Type: Eposter Presentation
Background
Splenic marginal zone lymphoma (SMZL) originates from the neoplastic transformation of mature B-lymphocytes. In most cases, B-lymphocytes infiltrate bone marrow (BM), suggesting a potential involvement of BM microenvironment in disease pathology.
Aims
To examine BM-derived mesenchymal stem/stromal cells (MSCs), since they comprise key components of the BM hematopoietic stroma, in order to investigate if they exhibit altered intrinsic properties in vitro or if they stimulate altered B- and T-cell immunomodulatory properties in SMZL patients compared to healthy controls.
Methods
BM MSCs were isolated and expanded in vitro from 12 SMZL patients and 11 healthy controls. MSCs were cultured for a total of 5 passages (P) and were phenotypically characterized by flow cytometry (FC). The colony forming unit-fibroblast (CFU-F) assay was used to estimate MSC frequency within the BM mononuclear cell (BMMC) fraction in P0 and their clonogenic capacity in P2. MSCs differentiation to adipocytes and osteoblasts was assessed by cytochemical stains. Their proliferative potential was evaluated by Methyl Triazolyl Tetrazolium assay. To assess the effect of SMZL MSCs on lymphocyte proliferation, B- and T-cells were immunomagnetically isolated (Miltenyi Biotec) from peripheral blood (PB) of normal individuals, labeled with carboxyfluorescein succinimidyl ester (Gibco) and subsequently cultured in the absence or presence of confluent layers of SMZL or normal MSCs, in the presence of activating factors. B cell apoptosis was evaluated via FC using 7-AAD staining, after co-culturing with SMZL or normal MSCs. Finally, B-cell chemotaxis was evaluated by monitoring the migration of isolated B-cells through a 5µm pore membrane in a transwell plate towards the confluent layers of either SMZL or normal MSCs.
Results
In vitro expanded MSCs from both groups were adherent cells with a spindle-shape morphology, which expressed CD29, CD73, CD90 and CD105 and were negative for the CD14, CD34 and CD45 cell surface markers. SMZL and Normal MSCs were similar in their differentiation to adipocytes and osteocytes as evidenced by Oil Red O and Alizarin Red staining, respectively. The frequency of MSCs within the BMMC compartment was significantly lower in patients as compared to healthy individuals (2.51 vs. 7.95 colonies per 105 ΒΜΜCs respectively; p=0.04) apparently due to the predominance of the lymphoma cells within patient BMMCs. SMZL MSCs in comparison to Normal MSCs at P2, displayed defective clonogenic potential (3.36 vs. 6.23 colonies respectively, p=0.03) and reduced proliferative potential (p<0.03).In co-culture experiments, SMZL and Normal MSCs showed no difference in their effect on normal B-cell or T-cell proliferation. However, SMZL MSCs produced a statistically significant advantage on B-cell survival as measured in B-cell apoptosis experiments. More specifically, 50±1.46% of B cells cultured in medium alone were apoptotic, while only 23±2.63% and 17±0.77% of B cells co-cultured with either Normal MSCs or SMZL MSCs were apoptotic (p=0.009 when comparing SMZL vs Normal MSCs). Finally the transwell migration assays showed that SMZL MSCs displayed a stronger chemotactic activity on isolated normal B-cells, in comparison to Normal MSCs, with the percentages of migrated B-cells being 34% and 22% respectively, (p=0.009).
Conclusion
Our data suggest that SMZL MSCs are intrinsically defective in terms of proliferative and clonogenic potential and moreover they exert distint anti-apoptotic and chemotactic effects on healthy-donor derived B cells. The impact of SMZL MSCs on the properties of patient derived peripheral B cells is currently under investigation.
Session topic: E-poster
Keyword(s): Mesenchymal stem cell, Microenvironment, Splenic marginal zone lymphoma
{{ help_message }}
{{filter}}