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Embryonic stem cell-derived B cells engraft in immunodeficient mice, recapitulating YS B lymphopoiesis
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
S103
3209 - CHARACTERIZATION OF MCL-1 ISOFORMS AND THEIR IMPACTS ON MAMMALIAN CELL GROWTH Hsin-Fang Yang-Yen, Hsiao-Ling Yeh, and Uan-I Chen Academia Sinica, Taipei, Taiwan
3211 - INTERFERON REGULATORY FACTOR-8 (IRF-8) REGULATES ERYTHROBLASTIC ISLAND MORPHOLOGY AND FUNCTION Jia Hao Yeo, Vicki Xie, Iain Campbell, and Stuart Fraser University of Sydney, Sydney/ Camperdown, New South Wales, Australia
Mcl-1 belongs to the anti-apoptotic subfamily of the Bcl-1 family protein, which has long been noticed to exist as two major isoforms with different gel-mobility. By extensive biochemical characterization, we identified that the slow mobility (SM) isoform is a full-length protein targeted to the outer membrane of mitochondria (MOM), whereas the fast-mobility (FM) isoform is an N-terminally truncated product generated inside the mitochondrial matrix via an MPP-mediated cleavage reaction. Interestingly, unlike the full-length protein located at MOM, the mitochondrial matrixlocalized isoform failed to interact with BH3-only proteins and manifested a greatly attenuated anti-apoptotic activity. A recently study by Perciavalle et al indicated that the matrix isoform plays a critical role in mitochondrial fusion, cristae ultrastructure, ATP production and respiration. However, its physiological function still remains elusive. To address this issue, we generated Mcl-1 knock-in mutant mice (the 3RG mutant) that are defective in producing the matrix isoform. Surprisingly, the 3RG mice were born with expected Mendelian frequency, and appeared to be developmentally normal. Moreover, in contrast to what was reported by Perciavalle et al. 3RG MEFs manifested similar growth rate, oxygen consumption and ATP production as the wild type cells, suggesting that the matrix isoform of Mcl-1 is dispensable under normal growth conditions. The impact of the matrix isoform on tumor growth in the lymphoid tissues is currently under investigation.
Adult mammalian erythropoiesis occurs within the bone marrow (BM) erythroid niches, termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters with a large central macrophage surrounded by smaller erythroid progenitors. We have developed a novel scanning electron microscopic assay analysing EBI morphology and structure to identify the molecular mechanisms regulating EBI function. Multicellular clusters were isolated from wild type bone marrow and their identity as EBIs confirmed using Ter119 antibody binding as detected by immunogold labelling. Using this protocol, we observed clustering of transferrin receptors around ‘‘nano-depressions’’ on reticulocytes. Furthermore, previously unreported pilus-like microstructures, which are distinct from structural features of dividing cells, were observed connecting between Ter119-expressing erythroblasts associating with an EBI central macrophage. We have termed these structures erythronanotubes. We examined mice lacking Interferon Regulatory Factor-8 (Irf8-/- mice), with morphological abnormalities in the microglia. These mutant mice showed profoundly reduction in BM erythroblast frequency compared to wild type BM. Our optimized SEM protocol also revealed an absence of classical EBIs and erythronanotubes. In contrast, we observed multi-cellular clusters with banded neutrophils interacting with central macrophages. F-actin, but not G-actin were absent in the Irf8-/- BM haematopoietic cells suggesting altered actin polymerization. These macrophages also lacked classical EBI macrophages surface markers (TIM-4, VCAM-1 and CD169) but not ICAM-1. EBI ex vivo reconstitution assays however revealed that the binding defect is primarily due to defects in Irf8-deficient erythroblasts. We are currently dissecting the defects in both erythroblasts and macrophages lacking Irf8 to identify the cause for EBI loss in these mutant mice
3210 - REPROGRAMMING ADULT MESENCHYMAL STEM CELLS INTO FUNCTIONAL HEMATOPOIETIC STEM CELLS THROUGH TRANSDETERMINATION - FROM MOLECULAR MECHANISM TO PRECISE THERAPY Qinwei Yin Program of Translational Medicine, The Military General Hospital of Beijing and Institute of Biophysics, CAS, Beijing, Zacatecas, China
3212 - EMBRYONIC STEM CELL-DERIVED B CELLS ENGRAFT IN IMMUNODEFICIENT MICE, RECAPITULATING YS B LYMPHOPOIESIS Yang Lin, Mervin C. Yoder, and Momoko Yoshimoto Indiana University, Indianapolis, Indiana, USA
Recently, ‘‘trans-determination’’ has attracted great controversy, mostly in regards to whether adult stem cells can colonize other tissues after transplantation. More importantly, how to generate large amounts of one type of functional stem cells from another type through a transdetermination process remained to be unsolved. Similarly, it is unclear whether mesenchymal stem cells (MSCs) can transdeterminate into hematopoietic stem cells (HSCs). Results: We report here that 1-3 X109 human adipose-derived MSCs (AD-MSCs) that are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could rapidly transdetermine into hematopoietic stem cells (CD49f+/CD133+/CD34+) and then their descending blood cells in vitro, after transfected with two small RNAs in the presence of special cytokines. The sRNAs were high-effectively delivered into MSCs by a novel peptide means. These adipose-derived HSCs (AD-HSCs) could form different types of hematopoietic colonies as nature-occurring CB-HSCs did. Upon transplantation into sublethally or lethally irradiated NOD/SCID mice, these AD-HSCs engrafted and differentiated into all hematopoietic lineages such as erythrocytes, lymphocytes, myelocytes and thrombocyte. Furthermore, we demonstrated the first evidence that the transdetermination of MSCs was induced by acetylation of histone proteins and activation of many transcriptional factors. As expected, these MSC-derived HSCs can not only save the life of lethally irradiated Balb/c mice, but also reconstitute hematopoietic function in patients with severe aplastic anemia. Conclusion: Our findings identify the sRNAs that dictates a directed transdifferentiation of MSCs toward HSCs, create a new source of HSCs used for the personalized treatment of precise defect such as severe aplastic anemia type II, and avoid bone marrow match and other many complications.
It has been reported that B cells are produced from embryonic stem cells (ESCs) when co-cultured with OP9 stromal cells. However, these ESC-derived B cells have not been well characterized; whether they belong to the B-1 or B-2 lineage and whether they are transplantable into immunodeficient mice. We have recently reported that the first wave of B lymphopoiesis occurring in the extra-embryonic yolk sac (YS) at the time prior to hematopoietic stem cell (HSC) emergence belongs to the B-1 cell lineage. HSC-independent B progenitor cells derived from E9.0 YS display a AA4.1+CD19+B220dim B-1 specific progenitor phenotype, are transplantable into immunodeficient mice, and secrete natural antibodies; all key features of B-1 cells. Since differentiation of ESC into blood cells recapitulates YS hematopoiesis, we hypothesized that B progenitor cells produced from mouse ESCs in vitro would belong to the B-1 cell lineage. We co-cultured ESCs with OP9 stromal cells and generated Flk1+ cells. Flk1+ cells were replated onto OP9 stromal cells, and after 9-12 days of co-culture, AA4.1+CD19+B220+ cells were detected by flow cytometry. We transplanted these cells into sublethally irradiated immunodeficient neonates. Four weeks after transplantation, donor-derived B cells were detected in the recipient peritoneal cavity, and displayed a B-1 cell phenotype. These ESC-derived B-1 cells were detected in the recipient peritoneal cavity up to 6 months after transplantation. Furthermore, these ESC-derived B-1 cells produced anti-phosphorylcholine antibodies detected by Elispot assay, showing the ability of these innate B-1 cells to produce natural antibodies. Thus, we have demonstrated that ESC-derived B cells engraft in vivo as functional B -1 cells secreting natural antibodies. These B cell developmental processes recapitulate B lymphopoiesis in the YS and might be utilized for innate immune cell therapy in the future.
S103
3209 - CHARACTERIZATION OF MCL-1 ISOFORMS AND THEIR IMPACTS ON MAMMALIAN CELL GROWTH Hsin-Fang Yang-Yen, Hsiao-Ling Yeh, and Uan-I Chen Academia Sinica, Taipei, Taiwan
3211 - INTERFERON REGULATORY FACTOR-8 (IRF-8) REGULATES ERYTHROBLASTIC ISLAND MORPHOLOGY AND FUNCTION Jia Hao Yeo, Vicki Xie, Iain Campbell, and Stuart Fraser University of Sydney, Sydney/ Camperdown, New South Wales, Australia
Mcl-1 belongs to the anti-apoptotic subfamily of the Bcl-1 family protein, which has long been noticed to exist as two major isoforms with different gel-mobility. By extensive biochemical characterization, we identified that the slow mobility (SM) isoform is a full-length protein targeted to the outer membrane of mitochondria (MOM), whereas the fast-mobility (FM) isoform is an N-terminally truncated product generated inside the mitochondrial matrix via an MPP-mediated cleavage reaction. Interestingly, unlike the full-length protein located at MOM, the mitochondrial matrixlocalized isoform failed to interact with BH3-only proteins and manifested a greatly attenuated anti-apoptotic activity. A recently study by Perciavalle et al indicated that the matrix isoform plays a critical role in mitochondrial fusion, cristae ultrastructure, ATP production and respiration. However, its physiological function still remains elusive. To address this issue, we generated Mcl-1 knock-in mutant mice (the 3RG mutant) that are defective in producing the matrix isoform. Surprisingly, the 3RG mice were born with expected Mendelian frequency, and appeared to be developmentally normal. Moreover, in contrast to what was reported by Perciavalle et al. 3RG MEFs manifested similar growth rate, oxygen consumption and ATP production as the wild type cells, suggesting that the matrix isoform of Mcl-1 is dispensable under normal growth conditions. The impact of the matrix isoform on tumor growth in the lymphoid tissues is currently under investigation.
Adult mammalian erythropoiesis occurs within the bone marrow (BM) erythroid niches, termed erythroblastic islands (EBIs). EBIs are multi-cellular clusters with a large central macrophage surrounded by smaller erythroid progenitors. We have developed a novel scanning electron microscopic assay analysing EBI morphology and structure to identify the molecular mechanisms regulating EBI function. Multicellular clusters were isolated from wild type bone marrow and their identity as EBIs confirmed using Ter119 antibody binding as detected by immunogold labelling. Using this protocol, we observed clustering of transferrin receptors around ‘‘nano-depressions’’ on reticulocytes. Furthermore, previously unreported pilus-like microstructures, which are distinct from structural features of dividing cells, were observed connecting between Ter119-expressing erythroblasts associating with an EBI central macrophage. We have termed these structures erythronanotubes. We examined mice lacking Interferon Regulatory Factor-8 (Irf8-/- mice), with morphological abnormalities in the microglia. These mutant mice showed profoundly reduction in BM erythroblast frequency compared to wild type BM. Our optimized SEM protocol also revealed an absence of classical EBIs and erythronanotubes. In contrast, we observed multi-cellular clusters with banded neutrophils interacting with central macrophages. F-actin, but not G-actin were absent in the Irf8-/- BM haematopoietic cells suggesting altered actin polymerization. These macrophages also lacked classical EBI macrophages surface markers (TIM-4, VCAM-1 and CD169) but not ICAM-1. EBI ex vivo reconstitution assays however revealed that the binding defect is primarily due to defects in Irf8-deficient erythroblasts. We are currently dissecting the defects in both erythroblasts and macrophages lacking Irf8 to identify the cause for EBI loss in these mutant mice
3210 - REPROGRAMMING ADULT MESENCHYMAL STEM CELLS INTO FUNCTIONAL HEMATOPOIETIC STEM CELLS THROUGH TRANSDETERMINATION - FROM MOLECULAR MECHANISM TO PRECISE THERAPY Qinwei Yin Program of Translational Medicine, The Military General Hospital of Beijing and Institute of Biophysics, CAS, Beijing, Zacatecas, China
3212 - EMBRYONIC STEM CELL-DERIVED B CELLS ENGRAFT IN IMMUNODEFICIENT MICE, RECAPITULATING YS B LYMPHOPOIESIS Yang Lin, Mervin C. Yoder, and Momoko Yoshimoto Indiana University, Indianapolis, Indiana, USA
Recently, ‘‘trans-determination’’ has attracted great controversy, mostly in regards to whether adult stem cells can colonize other tissues after transplantation. More importantly, how to generate large amounts of one type of functional stem cells from another type through a transdetermination process remained to be unsolved. Similarly, it is unclear whether mesenchymal stem cells (MSCs) can transdeterminate into hematopoietic stem cells (HSCs). Results: We report here that 1-3 X109 human adipose-derived MSCs (AD-MSCs) that are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could rapidly transdetermine into hematopoietic stem cells (CD49f+/CD133+/CD34+) and then their descending blood cells in vitro, after transfected with two small RNAs in the presence of special cytokines. The sRNAs were high-effectively delivered into MSCs by a novel peptide means. These adipose-derived HSCs (AD-HSCs) could form different types of hematopoietic colonies as nature-occurring CB-HSCs did. Upon transplantation into sublethally or lethally irradiated NOD/SCID mice, these AD-HSCs engrafted and differentiated into all hematopoietic lineages such as erythrocytes, lymphocytes, myelocytes and thrombocyte. Furthermore, we demonstrated the first evidence that the transdetermination of MSCs was induced by acetylation of histone proteins and activation of many transcriptional factors. As expected, these MSC-derived HSCs can not only save the life of lethally irradiated Balb/c mice, but also reconstitute hematopoietic function in patients with severe aplastic anemia. Conclusion: Our findings identify the sRNAs that dictates a directed transdifferentiation of MSCs toward HSCs, create a new source of HSCs used for the personalized treatment of precise defect such as severe aplastic anemia type II, and avoid bone marrow match and other many complications.
It has been reported that B cells are produced from embryonic stem cells (ESCs) when co-cultured with OP9 stromal cells. However, these ESC-derived B cells have not been well characterized; whether they belong to the B-1 or B-2 lineage and whether they are transplantable into immunodeficient mice. We have recently reported that the first wave of B lymphopoiesis occurring in the extra-embryonic yolk sac (YS) at the time prior to hematopoietic stem cell (HSC) emergence belongs to the B-1 cell lineage. HSC-independent B progenitor cells derived from E9.0 YS display a AA4.1+CD19+B220dim B-1 specific progenitor phenotype, are transplantable into immunodeficient mice, and secrete natural antibodies; all key features of B-1 cells. Since differentiation of ESC into blood cells recapitulates YS hematopoiesis, we hypothesized that B progenitor cells produced from mouse ESCs in vitro would belong to the B-1 cell lineage. We co-cultured ESCs with OP9 stromal cells and generated Flk1+ cells. Flk1+ cells were replated onto OP9 stromal cells, and after 9-12 days of co-culture, AA4.1+CD19+B220+ cells were detected by flow cytometry. We transplanted these cells into sublethally irradiated immunodeficient neonates. Four weeks after transplantation, donor-derived B cells were detected in the recipient peritoneal cavity, and displayed a B-1 cell phenotype. These ESC-derived B-1 cells were detected in the recipient peritoneal cavity up to 6 months after transplantation. Furthermore, these ESC-derived B-1 cells produced anti-phosphorylcholine antibodies detected by Elispot assay, showing the ability of these innate B-1 cells to produce natural antibodies. Thus, we have demonstrated that ESC-derived B cells engraft in vivo as functional B -1 cells secreting natural antibodies. These B cell developmental processes recapitulate B lymphopoiesis in the YS and might be utilized for innate immune cell therapy in the future.