MULTIPOTENT PROGENITOR CELLS AND ACQUIREMENT OF HEMATOPOIETIC CAPACITY IN THE BONE MARROW OF XENOPUS LAEVIS
(Abstract release date: 05/19/16)
EHA Library. Tanizaki Y. 06/09/16; 132682; E1133
Disclosure(s): There is no need to disclose actual financial value
Yuta Tanizaki
Contributions
Contributions
Abstract
Abstract: E1133
Type: Eposter Presentation
Background
Hematopoietic stem cells (HSCs) are characterized by their capacity for long-term repopulation and multilineage differentiation upon cytokine stimulation. Thrombopoietin (TPO) is one of the key regulators of HSC maintenance and platelet production. Previously, we cloned Xenopus laevis (X. laevis) TPO (xlTPO) and demonstrated that differentiation and proliferation in thrombocyte progenitors and hematopoietic progenitors are regulated by xlTPO. Unlike that of mammals, bone marrow (BM) of X. laevis is filled with adipose cells, and blood cells are mainly produced in the liver. However, functional contribution of fatty marrow for hematopoiesis is unclear, and multipotent hematopoietic progenitor is not yet identified in X. laevis.
Aims
In this study, we attempted to identify multipotent hematopoietic cells and evaluate the ability of hematopoiesis in fatty marrow in X. laevis.
Methods
The suspension cells in X. laevis were cultured in semi-solid culture system in the presence of xlTPO. Laparotomy was performed in X. laevis under anesthesia with MS222. The left liver was resected, and cultured with xlTPO for 24 days. The cultured cells were labeled by PKH26 and autologously transplanted by intracardiac injection. After 30 days, the X. laevis were killed and their right liver was analyzed by FACS. PKH26 labelled cultured cells were also injected into the blastocoels of X. laevis embryos at stage 8, which have no immune system. After 3 days, the labeled cells were analyzed by fluorescence microscope. For low-temperature exposure, cage containing X. laevis were transferred to an incubator set at 5°C for 12 days. For micro-computerized tomography (mCT) analysis, X. laevis were restrained in polystyrene foam restrainers, and the femurs were imaged by three-dimensional X-ray CT scan.
Results
Hepatic colonies stimulated by xlTPO could be cultured for more than 3 months, during which the cell number reached 1 × 106, indicating that the cells divided at least 20 times. These colonies expressed erythrocyte-, thrombocyte-, leukocyte-, and HSC-specific markers. Moreover, these cells differentiated to thrombocytes and leukocytes in the presence of splenic-conditioned media. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in presence of xlTPO, and cells labeled with PKH26 were autologously transplanted. After 30 days, PKH26-positive cells were detected in the sinusoids of liver and spleen. Flow cytometric analysis showed that the PKH26-positive cells displayed low forward (FSC) and side (SSC) scatter and had thin-layered cytoplasm and round nuclei, which are typical features of mammalian HSCs. These results indicated that xlTPO regulates proliferation of hematopoietic progenitors, which can be engrafted and differentiated to multiple lineages. To enrich multipotent hematopoietic cells, we generated anti-xlMPl monoclonal antibodies and showed that anti-thrombocyte antibody (T12)-/xlMpl+/FSClow population was enriched in high nuclear/cytoplasm ratio-hematopoietic progenitors, and the ratio of these cells to all hepatic cells was 0.28%. Surprisingly, T12-/xlMpl+/FSClow cells were identified in the liver, spleen, and BM in normal state. Although BM in X. laevis comprises mostly of adipocytes, large colonies induced by xlTPO were identified in the BM. The protein expression patterns in these cells overlapped with those in colonies derived from the liver, demonstrating that multipotent hematopoietic progenitors were localized in the BM. To explore the hematopoietic capacity in the BM, X. laevis were exposed to 5°C, which led to pancytopenia. After exposure to low temperature for 12 days, the numbers of erythrocyte and multipotent progenitors were increased. mCT revealed that femoral bone density was higher than that before exposure to 5°C, indicating change in the BM microenvironment.
Conclusion
In conclusion, multipotent hematopoietic progenitors in X. laevis are enriched in T12-/Mpl+/FSClow cells and these cells are mainly localized in the liver and BM. These findings suggested that T12-/Mpl+/FSClow cells in BM is dormant, and changes in the BM microenvironment by low-temperature stimulation induced proliferation of hematopoietic progenitors.
Session topic: E-poster
Keyword(s): Animal model, Bone microenvironment, Hematopoietic stem and progenitor cells, Thrombopoietin (TPO)
Type: Eposter Presentation
Background
Hematopoietic stem cells (HSCs) are characterized by their capacity for long-term repopulation and multilineage differentiation upon cytokine stimulation. Thrombopoietin (TPO) is one of the key regulators of HSC maintenance and platelet production. Previously, we cloned Xenopus laevis (X. laevis) TPO (xlTPO) and demonstrated that differentiation and proliferation in thrombocyte progenitors and hematopoietic progenitors are regulated by xlTPO. Unlike that of mammals, bone marrow (BM) of X. laevis is filled with adipose cells, and blood cells are mainly produced in the liver. However, functional contribution of fatty marrow for hematopoiesis is unclear, and multipotent hematopoietic progenitor is not yet identified in X. laevis.
Aims
In this study, we attempted to identify multipotent hematopoietic cells and evaluate the ability of hematopoiesis in fatty marrow in X. laevis.
Methods
The suspension cells in X. laevis were cultured in semi-solid culture system in the presence of xlTPO. Laparotomy was performed in X. laevis under anesthesia with MS222. The left liver was resected, and cultured with xlTPO for 24 days. The cultured cells were labeled by PKH26 and autologously transplanted by intracardiac injection. After 30 days, the X. laevis were killed and their right liver was analyzed by FACS. PKH26 labelled cultured cells were also injected into the blastocoels of X. laevis embryos at stage 8, which have no immune system. After 3 days, the labeled cells were analyzed by fluorescence microscope. For low-temperature exposure, cage containing X. laevis were transferred to an incubator set at 5°C for 12 days. For micro-computerized tomography (mCT) analysis, X. laevis were restrained in polystyrene foam restrainers, and the femurs were imaged by three-dimensional X-ray CT scan.
Results
Hepatic colonies stimulated by xlTPO could be cultured for more than 3 months, during which the cell number reached 1 × 106, indicating that the cells divided at least 20 times. These colonies expressed erythrocyte-, thrombocyte-, leukocyte-, and HSC-specific markers. Moreover, these cells differentiated to thrombocytes and leukocytes in the presence of splenic-conditioned media. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in presence of xlTPO, and cells labeled with PKH26 were autologously transplanted. After 30 days, PKH26-positive cells were detected in the sinusoids of liver and spleen. Flow cytometric analysis showed that the PKH26-positive cells displayed low forward (FSC) and side (SSC) scatter and had thin-layered cytoplasm and round nuclei, which are typical features of mammalian HSCs. These results indicated that xlTPO regulates proliferation of hematopoietic progenitors, which can be engrafted and differentiated to multiple lineages. To enrich multipotent hematopoietic cells, we generated anti-xlMPl monoclonal antibodies and showed that anti-thrombocyte antibody (T12)-/xlMpl+/FSClow population was enriched in high nuclear/cytoplasm ratio-hematopoietic progenitors, and the ratio of these cells to all hepatic cells was 0.28%. Surprisingly, T12-/xlMpl+/FSClow cells were identified in the liver, spleen, and BM in normal state. Although BM in X. laevis comprises mostly of adipocytes, large colonies induced by xlTPO were identified in the BM. The protein expression patterns in these cells overlapped with those in colonies derived from the liver, demonstrating that multipotent hematopoietic progenitors were localized in the BM. To explore the hematopoietic capacity in the BM, X. laevis were exposed to 5°C, which led to pancytopenia. After exposure to low temperature for 12 days, the numbers of erythrocyte and multipotent progenitors were increased. mCT revealed that femoral bone density was higher than that before exposure to 5°C, indicating change in the BM microenvironment.
Conclusion
In conclusion, multipotent hematopoietic progenitors in X. laevis are enriched in T12-/Mpl+/FSClow cells and these cells are mainly localized in the liver and BM. These findings suggested that T12-/Mpl+/FSClow cells in BM is dormant, and changes in the BM microenvironment by low-temperature stimulation induced proliferation of hematopoietic progenitors.
Session topic: E-poster
Keyword(s): Animal model, Bone microenvironment, Hematopoietic stem and progenitor cells, Thrombopoietin (TPO)
Abstract: E1133
Type: Eposter Presentation
Background
Hematopoietic stem cells (HSCs) are characterized by their capacity for long-term repopulation and multilineage differentiation upon cytokine stimulation. Thrombopoietin (TPO) is one of the key regulators of HSC maintenance and platelet production. Previously, we cloned Xenopus laevis (X. laevis) TPO (xlTPO) and demonstrated that differentiation and proliferation in thrombocyte progenitors and hematopoietic progenitors are regulated by xlTPO. Unlike that of mammals, bone marrow (BM) of X. laevis is filled with adipose cells, and blood cells are mainly produced in the liver. However, functional contribution of fatty marrow for hematopoiesis is unclear, and multipotent hematopoietic progenitor is not yet identified in X. laevis.
Aims
In this study, we attempted to identify multipotent hematopoietic cells and evaluate the ability of hematopoiesis in fatty marrow in X. laevis.
Methods
The suspension cells in X. laevis were cultured in semi-solid culture system in the presence of xlTPO. Laparotomy was performed in X. laevis under anesthesia with MS222. The left liver was resected, and cultured with xlTPO for 24 days. The cultured cells were labeled by PKH26 and autologously transplanted by intracardiac injection. After 30 days, the X. laevis were killed and their right liver was analyzed by FACS. PKH26 labelled cultured cells were also injected into the blastocoels of X. laevis embryos at stage 8, which have no immune system. After 3 days, the labeled cells were analyzed by fluorescence microscope. For low-temperature exposure, cage containing X. laevis were transferred to an incubator set at 5°C for 12 days. For micro-computerized tomography (mCT) analysis, X. laevis were restrained in polystyrene foam restrainers, and the femurs were imaged by three-dimensional X-ray CT scan.
Results
Hepatic colonies stimulated by xlTPO could be cultured for more than 3 months, during which the cell number reached 1 × 106, indicating that the cells divided at least 20 times. These colonies expressed erythrocyte-, thrombocyte-, leukocyte-, and HSC-specific markers. Moreover, these cells differentiated to thrombocytes and leukocytes in the presence of splenic-conditioned media. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in presence of xlTPO, and cells labeled with PKH26 were autologously transplanted. After 30 days, PKH26-positive cells were detected in the sinusoids of liver and spleen. Flow cytometric analysis showed that the PKH26-positive cells displayed low forward (FSC) and side (SSC) scatter and had thin-layered cytoplasm and round nuclei, which are typical features of mammalian HSCs. These results indicated that xlTPO regulates proliferation of hematopoietic progenitors, which can be engrafted and differentiated to multiple lineages. To enrich multipotent hematopoietic cells, we generated anti-xlMPl monoclonal antibodies and showed that anti-thrombocyte antibody (T12)-/xlMpl+/FSClow population was enriched in high nuclear/cytoplasm ratio-hematopoietic progenitors, and the ratio of these cells to all hepatic cells was 0.28%. Surprisingly, T12-/xlMpl+/FSClow cells were identified in the liver, spleen, and BM in normal state. Although BM in X. laevis comprises mostly of adipocytes, large colonies induced by xlTPO were identified in the BM. The protein expression patterns in these cells overlapped with those in colonies derived from the liver, demonstrating that multipotent hematopoietic progenitors were localized in the BM. To explore the hematopoietic capacity in the BM, X. laevis were exposed to 5°C, which led to pancytopenia. After exposure to low temperature for 12 days, the numbers of erythrocyte and multipotent progenitors were increased. mCT revealed that femoral bone density was higher than that before exposure to 5°C, indicating change in the BM microenvironment.
Conclusion
In conclusion, multipotent hematopoietic progenitors in X. laevis are enriched in T12-/Mpl+/FSClow cells and these cells are mainly localized in the liver and BM. These findings suggested that T12-/Mpl+/FSClow cells in BM is dormant, and changes in the BM microenvironment by low-temperature stimulation induced proliferation of hematopoietic progenitors.
Session topic: E-poster
Keyword(s): Animal model, Bone microenvironment, Hematopoietic stem and progenitor cells, Thrombopoietin (TPO)
Type: Eposter Presentation
Background
Hematopoietic stem cells (HSCs) are characterized by their capacity for long-term repopulation and multilineage differentiation upon cytokine stimulation. Thrombopoietin (TPO) is one of the key regulators of HSC maintenance and platelet production. Previously, we cloned Xenopus laevis (X. laevis) TPO (xlTPO) and demonstrated that differentiation and proliferation in thrombocyte progenitors and hematopoietic progenitors are regulated by xlTPO. Unlike that of mammals, bone marrow (BM) of X. laevis is filled with adipose cells, and blood cells are mainly produced in the liver. However, functional contribution of fatty marrow for hematopoiesis is unclear, and multipotent hematopoietic progenitor is not yet identified in X. laevis.
Aims
In this study, we attempted to identify multipotent hematopoietic cells and evaluate the ability of hematopoiesis in fatty marrow in X. laevis.
Methods
The suspension cells in X. laevis were cultured in semi-solid culture system in the presence of xlTPO. Laparotomy was performed in X. laevis under anesthesia with MS222. The left liver was resected, and cultured with xlTPO for 24 days. The cultured cells were labeled by PKH26 and autologously transplanted by intracardiac injection. After 30 days, the X. laevis were killed and their right liver was analyzed by FACS. PKH26 labelled cultured cells were also injected into the blastocoels of X. laevis embryos at stage 8, which have no immune system. After 3 days, the labeled cells were analyzed by fluorescence microscope. For low-temperature exposure, cage containing X. laevis were transferred to an incubator set at 5°C for 12 days. For micro-computerized tomography (mCT) analysis, X. laevis were restrained in polystyrene foam restrainers, and the femurs were imaged by three-dimensional X-ray CT scan.
Results
Hepatic colonies stimulated by xlTPO could be cultured for more than 3 months, during which the cell number reached 1 × 106, indicating that the cells divided at least 20 times. These colonies expressed erythrocyte-, thrombocyte-, leukocyte-, and HSC-specific markers. Moreover, these cells differentiated to thrombocytes and leukocytes in the presence of splenic-conditioned media. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in presence of xlTPO, and cells labeled with PKH26 were autologously transplanted. After 30 days, PKH26-positive cells were detected in the sinusoids of liver and spleen. Flow cytometric analysis showed that the PKH26-positive cells displayed low forward (FSC) and side (SSC) scatter and had thin-layered cytoplasm and round nuclei, which are typical features of mammalian HSCs. These results indicated that xlTPO regulates proliferation of hematopoietic progenitors, which can be engrafted and differentiated to multiple lineages. To enrich multipotent hematopoietic cells, we generated anti-xlMPl monoclonal antibodies and showed that anti-thrombocyte antibody (T12)-/xlMpl+/FSClow population was enriched in high nuclear/cytoplasm ratio-hematopoietic progenitors, and the ratio of these cells to all hepatic cells was 0.28%. Surprisingly, T12-/xlMpl+/FSClow cells were identified in the liver, spleen, and BM in normal state. Although BM in X. laevis comprises mostly of adipocytes, large colonies induced by xlTPO were identified in the BM. The protein expression patterns in these cells overlapped with those in colonies derived from the liver, demonstrating that multipotent hematopoietic progenitors were localized in the BM. To explore the hematopoietic capacity in the BM, X. laevis were exposed to 5°C, which led to pancytopenia. After exposure to low temperature for 12 days, the numbers of erythrocyte and multipotent progenitors were increased. mCT revealed that femoral bone density was higher than that before exposure to 5°C, indicating change in the BM microenvironment.
Conclusion
In conclusion, multipotent hematopoietic progenitors in X. laevis are enriched in T12-/Mpl+/FSClow cells and these cells are mainly localized in the liver and BM. These findings suggested that T12-/Mpl+/FSClow cells in BM is dormant, and changes in the BM microenvironment by low-temperature stimulation induced proliferation of hematopoietic progenitors.
Session topic: E-poster
Keyword(s): Animal model, Bone microenvironment, Hematopoietic stem and progenitor cells, Thrombopoietin (TPO)
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