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Declined presentation recombinant human thrombopoietin promotes hematopoietic reconstitution in mice
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Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3100 - HIGH FREQUENCY MUTATION OF 3’-UTR RELATED TO ABNORMAL EXPRESSION OF CD3z IN PATIENTS WITH APLASTIC ANEMIA Bo Li1, Su Fang1, Lixing Guo1, Ziwei Liao1, Mingjuan Wu2, Shaohua Chen1, Lijian Yang1, Xiang Lu3, and Yangqiu Li*,1,4 1 Institute of Hematology, Medical College, Jinan University, GuangZhou, GuangZhou, China; 2College of Life Science and Technology, Jinan University, Guangzhou, Guangzhou, China; 3Department of Hematology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China; 4Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, Guangzhou, China One of the main pathogenesis of aplastic anemia (AA) is abnormal T-cell immunity. Our previous study found that the expression level of CD3z was significant increased in AA. Therefore, in this study, to further understand the molecular mechanism of abnormalities of the TCR signaling pathway in AA, we further analyzed the expression and mutation of different isoforms of CD3z gene 3’ untranslated region (3’-UTR). The expression of different isoforms of CD3z 3’-UTR were analyzed from peripheral blood mononuclear cells in 29 healthy individuals and 45 patients with AA (35 newly diagnosed patients and 9 cases in remission) by RT-PCR. The mutations of CD3z 3’UTR were analyzed by cloning and nucleotide sequence analysis techniques. The wild typed CD3z 3’-UTR (WT) and alternative spliced CD3z 3’-UTR (AS) could be detected in all healthy individuals and AA cases in remission. Three types of CD3z 3’-UTR could be defined in untreated AA patients. 85.7% case of AA contained WT+AS+, 8.6% case contained WT+AS- and 5.7% cases contained WT-AS+ which has low CD3z expression level. There were different distribution characteristics, types of mutations in AA. Several new mutations were identified for the first time in the CD3z 3’-UTR in AA. Such as patients with 1184COG have significantly lower CD3z mRNA expression level. This is the first study describing characteristic of CD3z 3’-UTR in AA. The expression feature and mutations of CD3z 3’-UTR may be affect CD3z mRNA expression level in some AA patients. This study was sponsored by grants from the National Natural Science Foundation of China (#81370605 and #81460026), a China Post-doctoral Science Foundation funded project (#20070410840), the Natural Science Foundation of Guangdong Province (#S2012010008794), and the Fundamental Research Funds for the Central Universities (#21612425).
3102 - LOCALIZATION AND CHARACTERIZATION OF HEMATOPOIETIC CELLS IN THE MOUSE EMBRYONIC HEAD Zhuan Li, Chris S. Vink, Samanta Mariani, and Elaine Dzierzak University of Edinburgh, Edinburgh, United Kingdom The midgestation mouse head harbors hematopoietic stem cells (HSCs) at the same time HSCs are detected in the aorta, gonads, mesonephros region (AGM). Within the complex structure of the embryonic head, the localization and phenotype of HSCs and hematopoietic progenitor cells (HPCs) remains unknown. Since the Ly6a(Sca1)GFP transgene is a reporter for all AGM HSCs and some hemogenic endothelial cells (HEC), we examined these embryos for head HSC, HEC and HPC. Wholemount staining reveals some GFP+ cells in E10.5 head vasculature. By FACS analysis, 0.6% of E11.5 head cells are GFP+. To examine whether head HPC/HSC are enriched in the Ly6aGFP+ population, we sorted E11.5-E12.5 head cells and performed functional assays. Preliminary data show that head HSCs are GFP+. When we subdissected the head into forebrain (FB), midbrain (MB), and hindbrain with brachial arches (HBA) to localize HPCs, we found erythro-myeloid progenitor cells in GFP+ and GFP- fractions of all three brain regions at E10-11.5. Interestingly, in OP9 co-cutures, B cell potential was mostly observed in CD31+GFP+ of the three subregions. These results suggest that head HSCs (Ly6aGFP) are phenotypically and functionally similar to AGM HSCs. We are continuing to identify the precise head subregion(s) in which HSCs occur, examine the hemogenic potential of the head vasculature, and also the pathways affecting their development.
3101 - FUNCTIONAL PERTURBATIONS IN MESENCHYMAL STROMAL CELLS FROM PATIENTS WITH ACUTE MYELOID LEUKEMIA AND FOLLOWING HIGH-DOSE IRRADIATION Y. Le1, P. Chandran2, R. Richardson1, and D. Allan2 1 Canadian Nuclear Laboratories, Chalk River, Ontario, Canada; 2Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
3103 - DECLINED PRESENTATION RECOMBINANT HUMAN THROMBOPOIETIN PROMOTES HEMATOPOIETIC RECONSTITUTION IN MICE Yanhua Li1, Chao Wang2, Bowen Zhang1, Sihan Wang1, Lijuan He1, and Xuetao Pei1 1 Beijing Institute of transfusion Medicine, Beijing, China; 2Shenyang Sunshine Pharmaceutical Co., Ltd., Shenyang, China
Background: Total body irradiation-based hematopoietic stem cell transplantation (TBI-HSCT) is a promising strategy for treating acute myeloid leukemia (AML). Unfortunately, this approach is often insufficient to eradicate leukemia and prevent its relapse. While HSCT supplies donor-derived HSCs, the hematopoietic niche remains patient-derived and undergoes damage due to TBI. The key cellular component of the marrow niche is the mesenchymal stromal cell (MSC). Importance of MSCs in normal and malignant hematopoiesis has been previously demonstrated, however, the extent to which MSC function is perturbed following high-dose irradiation and in patients with AML remains unknown. We hypothesize that MSC function is perturbed in AML and following TBI resulting in altered microenvironment that is permissive to leukemia relapse. Methods: We have compared functional properties of bone marrow MSCs from newly diagnosed AML patients and normal donors and tested the effects of high dose radiation on MSC function. Results: Flow cytometric analyses showed normal immunophenotype and viability of AML-derived and irradiated MSCs. However, there was a significant decrease in proliferation (S phase) of both irradiated and AML-MSCs with a concomitant increase in proportion of G1/G0 arrested cells. Additionally, both irradiated and AML-MSCs exhibited increased adipogenic and inhibited osteogenic differentiation. Co-culture of UCB-CD34+ cells with adipoMSCs vs. osteo-MSCs resulted in increased expansion and decreased hematopoietic colony forming capacity of CD34+ cells consistent with HSPC pool exhaustion. Conclusions: Taken together, AML patient-derived and irradiated MSCs exhibited similar functional changes that are characterized by decreased cell proliferation, cell cycle arrest and a bias toward adipogenesis. Adipo-MSCs resulted in depletion of normal in vitro hematopoietic potential of CD34+ cells suggestive of abnormal microenvironment. Thus, alternative transplant strategies are needed that restore the health of bone marrow microenvironment in patients with AML and following TBI.
Recombinant human TPO (rHuTPO, TIPAO) is a clinical drug for promoting megakaryocyte and platelet generation. Here, we reported that rHuTPO could have a therapeutic role in promoting hematopoietic reconstitution. Given that hematopoietic cytokines exhibited little effect on BM reconstruction in the mice after lethal irradiation, we transplanted 5105 or 1106 bone marrow MNCs into the recipient mice after 9.5 Gy exposure. These mice were subsequently administered with rHuTPO (25mg/kg) or PBS for 14 consecutive days. We found that 100% of the mice that received 1106 BM cells and 14 consecutive days of TPO treatment survived. In contrast, 60% of the recipients in the control treatment group survived. The transplantation of 5105 bone marrow cells rescued 70% of the recipient mice that received TPO treatment. A lower survival rate was observed in the mice that received 5105 bone marrow cells and PBS treatment. These results suggested that rHuTPO enhanced the survival of lethally irradiated mice following bone marrow transplantation. To further investigate the role of rHuTPO after HSC transplantation, donor bone marrow cells from b-actin-luciferase transgenic mice were transplanted into lethally irradiated mice. The recipient mice were administered with rHuTPO (25mg/kg) or PBS (control) daily for 14 days. The in vivo luciferase imaging results revealed that the donor bone marrow cells were more thoroughly engrafted in the rHuTPOtreated recipient mice than in the PBS-treated mice, particularly at 0.5-3 months after transplantation. Importantly, rHuTPO treatment significantly increased transplanted hematopoietic cell engraftment in the bone marrow and spleen of the recipients. The rHuTPO treatment group also exhibited a significantly increased frequency of donor CD45.2+ LSK cells in the bone marrow. These data suggested that rHuTPO enhanced HSC expansion and engraftment in lethally irradiated mice.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3100 - HIGH FREQUENCY MUTATION OF 3’-UTR RELATED TO ABNORMAL EXPRESSION OF CD3z IN PATIENTS WITH APLASTIC ANEMIA Bo Li1, Su Fang1, Lixing Guo1, Ziwei Liao1, Mingjuan Wu2, Shaohua Chen1, Lijian Yang1, Xiang Lu3, and Yangqiu Li*,1,4 1 Institute of Hematology, Medical College, Jinan University, GuangZhou, GuangZhou, China; 2College of Life Science and Technology, Jinan University, Guangzhou, Guangzhou, China; 3Department of Hematology, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, China; 4Key Laboratory for Regenerative Medicine of Ministry of Education, Jinan University, Guangzhou, Guangzhou, China One of the main pathogenesis of aplastic anemia (AA) is abnormal T-cell immunity. Our previous study found that the expression level of CD3z was significant increased in AA. Therefore, in this study, to further understand the molecular mechanism of abnormalities of the TCR signaling pathway in AA, we further analyzed the expression and mutation of different isoforms of CD3z gene 3’ untranslated region (3’-UTR). The expression of different isoforms of CD3z 3’-UTR were analyzed from peripheral blood mononuclear cells in 29 healthy individuals and 45 patients with AA (35 newly diagnosed patients and 9 cases in remission) by RT-PCR. The mutations of CD3z 3’UTR were analyzed by cloning and nucleotide sequence analysis techniques. The wild typed CD3z 3’-UTR (WT) and alternative spliced CD3z 3’-UTR (AS) could be detected in all healthy individuals and AA cases in remission. Three types of CD3z 3’-UTR could be defined in untreated AA patients. 85.7% case of AA contained WT+AS+, 8.6% case contained WT+AS- and 5.7% cases contained WT-AS+ which has low CD3z expression level. There were different distribution characteristics, types of mutations in AA. Several new mutations were identified for the first time in the CD3z 3’-UTR in AA. Such as patients with 1184COG have significantly lower CD3z mRNA expression level. This is the first study describing characteristic of CD3z 3’-UTR in AA. The expression feature and mutations of CD3z 3’-UTR may be affect CD3z mRNA expression level in some AA patients. This study was sponsored by grants from the National Natural Science Foundation of China (#81370605 and #81460026), a China Post-doctoral Science Foundation funded project (#20070410840), the Natural Science Foundation of Guangdong Province (#S2012010008794), and the Fundamental Research Funds for the Central Universities (#21612425).
3102 - LOCALIZATION AND CHARACTERIZATION OF HEMATOPOIETIC CELLS IN THE MOUSE EMBRYONIC HEAD Zhuan Li, Chris S. Vink, Samanta Mariani, and Elaine Dzierzak University of Edinburgh, Edinburgh, United Kingdom The midgestation mouse head harbors hematopoietic stem cells (HSCs) at the same time HSCs are detected in the aorta, gonads, mesonephros region (AGM). Within the complex structure of the embryonic head, the localization and phenotype of HSCs and hematopoietic progenitor cells (HPCs) remains unknown. Since the Ly6a(Sca1)GFP transgene is a reporter for all AGM HSCs and some hemogenic endothelial cells (HEC), we examined these embryos for head HSC, HEC and HPC. Wholemount staining reveals some GFP+ cells in E10.5 head vasculature. By FACS analysis, 0.6% of E11.5 head cells are GFP+. To examine whether head HPC/HSC are enriched in the Ly6aGFP+ population, we sorted E11.5-E12.5 head cells and performed functional assays. Preliminary data show that head HSCs are GFP+. When we subdissected the head into forebrain (FB), midbrain (MB), and hindbrain with brachial arches (HBA) to localize HPCs, we found erythro-myeloid progenitor cells in GFP+ and GFP- fractions of all three brain regions at E10-11.5. Interestingly, in OP9 co-cutures, B cell potential was mostly observed in CD31+GFP+ of the three subregions. These results suggest that head HSCs (Ly6aGFP) are phenotypically and functionally similar to AGM HSCs. We are continuing to identify the precise head subregion(s) in which HSCs occur, examine the hemogenic potential of the head vasculature, and also the pathways affecting their development.
3101 - FUNCTIONAL PERTURBATIONS IN MESENCHYMAL STROMAL CELLS FROM PATIENTS WITH ACUTE MYELOID LEUKEMIA AND FOLLOWING HIGH-DOSE IRRADIATION Y. Le1, P. Chandran2, R. Richardson1, and D. Allan2 1 Canadian Nuclear Laboratories, Chalk River, Ontario, Canada; 2Ottawa Hospital Research Institute, Ottawa, Ontario, Canada
3103 - DECLINED PRESENTATION RECOMBINANT HUMAN THROMBOPOIETIN PROMOTES HEMATOPOIETIC RECONSTITUTION IN MICE Yanhua Li1, Chao Wang2, Bowen Zhang1, Sihan Wang1, Lijuan He1, and Xuetao Pei1 1 Beijing Institute of transfusion Medicine, Beijing, China; 2Shenyang Sunshine Pharmaceutical Co., Ltd., Shenyang, China
Background: Total body irradiation-based hematopoietic stem cell transplantation (TBI-HSCT) is a promising strategy for treating acute myeloid leukemia (AML). Unfortunately, this approach is often insufficient to eradicate leukemia and prevent its relapse. While HSCT supplies donor-derived HSCs, the hematopoietic niche remains patient-derived and undergoes damage due to TBI. The key cellular component of the marrow niche is the mesenchymal stromal cell (MSC). Importance of MSCs in normal and malignant hematopoiesis has been previously demonstrated, however, the extent to which MSC function is perturbed following high-dose irradiation and in patients with AML remains unknown. We hypothesize that MSC function is perturbed in AML and following TBI resulting in altered microenvironment that is permissive to leukemia relapse. Methods: We have compared functional properties of bone marrow MSCs from newly diagnosed AML patients and normal donors and tested the effects of high dose radiation on MSC function. Results: Flow cytometric analyses showed normal immunophenotype and viability of AML-derived and irradiated MSCs. However, there was a significant decrease in proliferation (S phase) of both irradiated and AML-MSCs with a concomitant increase in proportion of G1/G0 arrested cells. Additionally, both irradiated and AML-MSCs exhibited increased adipogenic and inhibited osteogenic differentiation. Co-culture of UCB-CD34+ cells with adipoMSCs vs. osteo-MSCs resulted in increased expansion and decreased hematopoietic colony forming capacity of CD34+ cells consistent with HSPC pool exhaustion. Conclusions: Taken together, AML patient-derived and irradiated MSCs exhibited similar functional changes that are characterized by decreased cell proliferation, cell cycle arrest and a bias toward adipogenesis. Adipo-MSCs resulted in depletion of normal in vitro hematopoietic potential of CD34+ cells suggestive of abnormal microenvironment. Thus, alternative transplant strategies are needed that restore the health of bone marrow microenvironment in patients with AML and following TBI.
Recombinant human TPO (rHuTPO, TIPAO) is a clinical drug for promoting megakaryocyte and platelet generation. Here, we reported that rHuTPO could have a therapeutic role in promoting hematopoietic reconstitution. Given that hematopoietic cytokines exhibited little effect on BM reconstruction in the mice after lethal irradiation, we transplanted 5105 or 1106 bone marrow MNCs into the recipient mice after 9.5 Gy exposure. These mice were subsequently administered with rHuTPO (25mg/kg) or PBS for 14 consecutive days. We found that 100% of the mice that received 1106 BM cells and 14 consecutive days of TPO treatment survived. In contrast, 60% of the recipients in the control treatment group survived. The transplantation of 5105 bone marrow cells rescued 70% of the recipient mice that received TPO treatment. A lower survival rate was observed in the mice that received 5105 bone marrow cells and PBS treatment. These results suggested that rHuTPO enhanced the survival of lethally irradiated mice following bone marrow transplantation. To further investigate the role of rHuTPO after HSC transplantation, donor bone marrow cells from b-actin-luciferase transgenic mice were transplanted into lethally irradiated mice. The recipient mice were administered with rHuTPO (25mg/kg) or PBS (control) daily for 14 days. The in vivo luciferase imaging results revealed that the donor bone marrow cells were more thoroughly engrafted in the rHuTPOtreated recipient mice than in the PBS-treated mice, particularly at 0.5-3 months after transplantation. Importantly, rHuTPO treatment significantly increased transplanted hematopoietic cell engraftment in the bone marrow and spleen of the recipients. The rHuTPO treatment group also exhibited a significantly increased frequency of donor CD45.2+ LSK cells in the bone marrow. These data suggested that rHuTPO enhanced HSC expansion and engraftment in lethally irradiated mice.