THE APC/C COACTIVATOR CDH1 CONTROLS MAINTENANCE OF THE HEMATOPOIETIC STEM CELL POOL IN VIVO
(Abstract release date: 05/19/16)
EHA Library. Kreutmair S. 06/09/16; 132674; E1125
Ms. Stefanie Kreutmair
Contributions
Contributions
Abstract
Abstract: E1125
Type: Eposter Presentation
Background
Cdh1 is a co-activator of the anaphase-promoting complex (APC/C) and highly active during G1 to target cell cycle proteins for proteasomal degradation thereby regulating cell proliferation. Recently, evidence accumulates, that Cdh1 may play a role in proliferation, self-renewal and multilineage differentiation of hematopoietic stem cells (HSCs).
Aims
Our objective was to examine the functional role of Cdh1 in the hematopoietic compartment in vivo.
Methods
We used a murine transplantation model in combination with targeted genetic RNAi approach for Cdh1 deletion in a competitive transplantation (TX) assay. Therefore, 5-FU enriched murine bone marrow cells (BMCs) were lineage depleted and retrovirally infected either with pLMPmiRCtrl or pLMPmiRCDH1. Primary BMCs and equal proportions of stem and progenitor cells with significant Cdh1 knockdown were transplanted in competition with non-infected wildtype BMCs into lethally irradiated wildtype recipients.
Results
Peripheral blood (PB) chimerism of recipient mice revealed a significant higher ratio of EGFP+ leukocytes in recipients of pLMPmiRCDH1 BMCs compared to controls (p=0.009). Moreover, spleen and BM chimerism showed significantly enhanced repopulation of Cdh1 knockdown cells at week 3 compared to pLMPmiRCtrl BMC recipients (p=0.004). Furthermore, Cdh1 knockdown leads to a significant decrease of apoptotic cells compared to control BMCs. Additionally, a highly significant decrease in the Sub-G1 cell population in Cdh1 knockdown BMC recipients confirms enhanced viability of Cdh1 knockdown cells. In contrast to apoptosis alterations, no obvious difference in cell cycling could be monitored between sorted EGFP+ pLMPmiRCtrl or pLMPmiRCDH1 infected BMCs of recipient mice 3 and 8 weeks after TX by dynamic 2-dimensional EdU/FxCycle violet assays. Interestingly, the difference in chimerism vanished during the course of the TX assay. To evaluate differences in the repopulation capacity of Cdh1 knockdown and control BMCs in transplanted animals, FACS analysis were performed and showed a significant reduction of LK- (lineage-, cKit+, Sca1-) and LSK- (lineage-, cKit+, Sca1+) cell frequency within pLMPmiRCDH1 BMC recipient mice compared to controls at the end of TX indicating that Cdh1 knockdown leads to an initial increase in repopulation capacity followed by exhaustion of hematopoietic stem and progenitor cells in a competing situation. To examine whether Cdh1 modulates lineage differentiation of HSCs we performed FACS analyses and detected a significantly higher proportion of B-cells in recipient mice transplanted with pLMPmiRCDH1 BMCs compared to control animals. Analyses of the myeloid compartment displayed differences with reduced levels of granulocytes as well as monocytes in the PB of Cdh1 knockdown transplanted BMCs compared to the pLMPmiRCtrl group. T-cells were not affected significantly by Cdh1 knockdown, at least in this short transplantation setting.
Conclusion
Taken together, Cdh1 knockdown leads to enhanced viability as a consequence of less susceptibility to apoptosis in vitro and in vivo, suggesting that Cdh1 plays an important role in maintaining the stem cell numbers within the HSC pool. Furthermore, Cdh1 knockdown regulates lineage differentiation in favor to the B-cell differentiation, whereas the myeloid lineage is repressed in Cdh1 knockdown cells in competitive transplantation experiments.
Session topic: E-poster
Keyword(s): Differentiation, Hematopoiesis, Hematopoietic stem and progenitor cells, Self-renewal
Type: Eposter Presentation
Background
Cdh1 is a co-activator of the anaphase-promoting complex (APC/C) and highly active during G1 to target cell cycle proteins for proteasomal degradation thereby regulating cell proliferation. Recently, evidence accumulates, that Cdh1 may play a role in proliferation, self-renewal and multilineage differentiation of hematopoietic stem cells (HSCs).
Aims
Our objective was to examine the functional role of Cdh1 in the hematopoietic compartment in vivo.
Methods
We used a murine transplantation model in combination with targeted genetic RNAi approach for Cdh1 deletion in a competitive transplantation (TX) assay. Therefore, 5-FU enriched murine bone marrow cells (BMCs) were lineage depleted and retrovirally infected either with pLMPmiRCtrl or pLMPmiRCDH1. Primary BMCs and equal proportions of stem and progenitor cells with significant Cdh1 knockdown were transplanted in competition with non-infected wildtype BMCs into lethally irradiated wildtype recipients.
Results
Peripheral blood (PB) chimerism of recipient mice revealed a significant higher ratio of EGFP+ leukocytes in recipients of pLMPmiRCDH1 BMCs compared to controls (p=0.009). Moreover, spleen and BM chimerism showed significantly enhanced repopulation of Cdh1 knockdown cells at week 3 compared to pLMPmiRCtrl BMC recipients (p=0.004). Furthermore, Cdh1 knockdown leads to a significant decrease of apoptotic cells compared to control BMCs. Additionally, a highly significant decrease in the Sub-G1 cell population in Cdh1 knockdown BMC recipients confirms enhanced viability of Cdh1 knockdown cells. In contrast to apoptosis alterations, no obvious difference in cell cycling could be monitored between sorted EGFP+ pLMPmiRCtrl or pLMPmiRCDH1 infected BMCs of recipient mice 3 and 8 weeks after TX by dynamic 2-dimensional EdU/FxCycle violet assays. Interestingly, the difference in chimerism vanished during the course of the TX assay. To evaluate differences in the repopulation capacity of Cdh1 knockdown and control BMCs in transplanted animals, FACS analysis were performed and showed a significant reduction of LK- (lineage-, cKit+, Sca1-) and LSK- (lineage-, cKit+, Sca1+) cell frequency within pLMPmiRCDH1 BMC recipient mice compared to controls at the end of TX indicating that Cdh1 knockdown leads to an initial increase in repopulation capacity followed by exhaustion of hematopoietic stem and progenitor cells in a competing situation. To examine whether Cdh1 modulates lineage differentiation of HSCs we performed FACS analyses and detected a significantly higher proportion of B-cells in recipient mice transplanted with pLMPmiRCDH1 BMCs compared to control animals. Analyses of the myeloid compartment displayed differences with reduced levels of granulocytes as well as monocytes in the PB of Cdh1 knockdown transplanted BMCs compared to the pLMPmiRCtrl group. T-cells were not affected significantly by Cdh1 knockdown, at least in this short transplantation setting.
Conclusion
Taken together, Cdh1 knockdown leads to enhanced viability as a consequence of less susceptibility to apoptosis in vitro and in vivo, suggesting that Cdh1 plays an important role in maintaining the stem cell numbers within the HSC pool. Furthermore, Cdh1 knockdown regulates lineage differentiation in favor to the B-cell differentiation, whereas the myeloid lineage is repressed in Cdh1 knockdown cells in competitive transplantation experiments.
Session topic: E-poster
Keyword(s): Differentiation, Hematopoiesis, Hematopoietic stem and progenitor cells, Self-renewal
Abstract: E1125
Type: Eposter Presentation
Background
Cdh1 is a co-activator of the anaphase-promoting complex (APC/C) and highly active during G1 to target cell cycle proteins for proteasomal degradation thereby regulating cell proliferation. Recently, evidence accumulates, that Cdh1 may play a role in proliferation, self-renewal and multilineage differentiation of hematopoietic stem cells (HSCs).
Aims
Our objective was to examine the functional role of Cdh1 in the hematopoietic compartment in vivo.
Methods
We used a murine transplantation model in combination with targeted genetic RNAi approach for Cdh1 deletion in a competitive transplantation (TX) assay. Therefore, 5-FU enriched murine bone marrow cells (BMCs) were lineage depleted and retrovirally infected either with pLMPmiRCtrl or pLMPmiRCDH1. Primary BMCs and equal proportions of stem and progenitor cells with significant Cdh1 knockdown were transplanted in competition with non-infected wildtype BMCs into lethally irradiated wildtype recipients.
Results
Peripheral blood (PB) chimerism of recipient mice revealed a significant higher ratio of EGFP+ leukocytes in recipients of pLMPmiRCDH1 BMCs compared to controls (p=0.009). Moreover, spleen and BM chimerism showed significantly enhanced repopulation of Cdh1 knockdown cells at week 3 compared to pLMPmiRCtrl BMC recipients (p=0.004). Furthermore, Cdh1 knockdown leads to a significant decrease of apoptotic cells compared to control BMCs. Additionally, a highly significant decrease in the Sub-G1 cell population in Cdh1 knockdown BMC recipients confirms enhanced viability of Cdh1 knockdown cells. In contrast to apoptosis alterations, no obvious difference in cell cycling could be monitored between sorted EGFP+ pLMPmiRCtrl or pLMPmiRCDH1 infected BMCs of recipient mice 3 and 8 weeks after TX by dynamic 2-dimensional EdU/FxCycle violet assays. Interestingly, the difference in chimerism vanished during the course of the TX assay. To evaluate differences in the repopulation capacity of Cdh1 knockdown and control BMCs in transplanted animals, FACS analysis were performed and showed a significant reduction of LK- (lineage-, cKit+, Sca1-) and LSK- (lineage-, cKit+, Sca1+) cell frequency within pLMPmiRCDH1 BMC recipient mice compared to controls at the end of TX indicating that Cdh1 knockdown leads to an initial increase in repopulation capacity followed by exhaustion of hematopoietic stem and progenitor cells in a competing situation. To examine whether Cdh1 modulates lineage differentiation of HSCs we performed FACS analyses and detected a significantly higher proportion of B-cells in recipient mice transplanted with pLMPmiRCDH1 BMCs compared to control animals. Analyses of the myeloid compartment displayed differences with reduced levels of granulocytes as well as monocytes in the PB of Cdh1 knockdown transplanted BMCs compared to the pLMPmiRCtrl group. T-cells were not affected significantly by Cdh1 knockdown, at least in this short transplantation setting.
Conclusion
Taken together, Cdh1 knockdown leads to enhanced viability as a consequence of less susceptibility to apoptosis in vitro and in vivo, suggesting that Cdh1 plays an important role in maintaining the stem cell numbers within the HSC pool. Furthermore, Cdh1 knockdown regulates lineage differentiation in favor to the B-cell differentiation, whereas the myeloid lineage is repressed in Cdh1 knockdown cells in competitive transplantation experiments.
Session topic: E-poster
Keyword(s): Differentiation, Hematopoiesis, Hematopoietic stem and progenitor cells, Self-renewal
Type: Eposter Presentation
Background
Cdh1 is a co-activator of the anaphase-promoting complex (APC/C) and highly active during G1 to target cell cycle proteins for proteasomal degradation thereby regulating cell proliferation. Recently, evidence accumulates, that Cdh1 may play a role in proliferation, self-renewal and multilineage differentiation of hematopoietic stem cells (HSCs).
Aims
Our objective was to examine the functional role of Cdh1 in the hematopoietic compartment in vivo.
Methods
We used a murine transplantation model in combination with targeted genetic RNAi approach for Cdh1 deletion in a competitive transplantation (TX) assay. Therefore, 5-FU enriched murine bone marrow cells (BMCs) were lineage depleted and retrovirally infected either with pLMPmiRCtrl or pLMPmiRCDH1. Primary BMCs and equal proportions of stem and progenitor cells with significant Cdh1 knockdown were transplanted in competition with non-infected wildtype BMCs into lethally irradiated wildtype recipients.
Results
Peripheral blood (PB) chimerism of recipient mice revealed a significant higher ratio of EGFP+ leukocytes in recipients of pLMPmiRCDH1 BMCs compared to controls (p=0.009). Moreover, spleen and BM chimerism showed significantly enhanced repopulation of Cdh1 knockdown cells at week 3 compared to pLMPmiRCtrl BMC recipients (p=0.004). Furthermore, Cdh1 knockdown leads to a significant decrease of apoptotic cells compared to control BMCs. Additionally, a highly significant decrease in the Sub-G1 cell population in Cdh1 knockdown BMC recipients confirms enhanced viability of Cdh1 knockdown cells. In contrast to apoptosis alterations, no obvious difference in cell cycling could be monitored between sorted EGFP+ pLMPmiRCtrl or pLMPmiRCDH1 infected BMCs of recipient mice 3 and 8 weeks after TX by dynamic 2-dimensional EdU/FxCycle violet assays. Interestingly, the difference in chimerism vanished during the course of the TX assay. To evaluate differences in the repopulation capacity of Cdh1 knockdown and control BMCs in transplanted animals, FACS analysis were performed and showed a significant reduction of LK- (lineage-, cKit+, Sca1-) and LSK- (lineage-, cKit+, Sca1+) cell frequency within pLMPmiRCDH1 BMC recipient mice compared to controls at the end of TX indicating that Cdh1 knockdown leads to an initial increase in repopulation capacity followed by exhaustion of hematopoietic stem and progenitor cells in a competing situation. To examine whether Cdh1 modulates lineage differentiation of HSCs we performed FACS analyses and detected a significantly higher proportion of B-cells in recipient mice transplanted with pLMPmiRCDH1 BMCs compared to control animals. Analyses of the myeloid compartment displayed differences with reduced levels of granulocytes as well as monocytes in the PB of Cdh1 knockdown transplanted BMCs compared to the pLMPmiRCtrl group. T-cells were not affected significantly by Cdh1 knockdown, at least in this short transplantation setting.
Conclusion
Taken together, Cdh1 knockdown leads to enhanced viability as a consequence of less susceptibility to apoptosis in vitro and in vivo, suggesting that Cdh1 plays an important role in maintaining the stem cell numbers within the HSC pool. Furthermore, Cdh1 knockdown regulates lineage differentiation in favor to the B-cell differentiation, whereas the myeloid lineage is repressed in Cdh1 knockdown cells in competitive transplantation experiments.
Session topic: E-poster
Keyword(s): Differentiation, Hematopoiesis, Hematopoietic stem and progenitor cells, Self-renewal
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