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Role of spred1 in the regulation of self-renewal and aging of hematopoietic stem cells
S96
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3180 - MAINTENANCE AND EXPANSION OF HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS IN THE PRESENCE OF DEFINED FACTORS ALONE Kazuhiro Sudo1, Satoshi Yamazaki2, Hiromitsu Nakauchi2, and Yukio Nakamura1 1 RIKEN BRC, Tsukuba, Ibaraki, Japan; 2University of Tokyo, Tokyo, Japan Human hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) or umbilical cord blood (CB) are very rare cell populations. Especially the number of HSPCs in CB derived from one donor is quite limited. Ex vivo expansion is thought to be a promising procedure to produce the sufficient number of human HSPC for clinical transplantations. Studies about ex vivo expansion of HSPCs have been reported. In many cases, animal-derived components, such as fetal bovine serum (FBS) and bovine serum albumin fraction V (BSA-V), are added to the culture. It is well known that different lot of BSA-V give different effects to HSPC proliferation. Most recently, we found that mouse hematopoietic stem cells (HSCs) cultivated in different lot of BSA-V-contained culture condition have different ability to reconstitute irradiated mouse hematopoiesis even if all BSA-V give the comparable level of cell proliferation level. This result strongly suggests that addition of animalderived component(s) to HSC-culture provoke the instability. In addition to the instability of experimental data, animal-derived components should be excluded as much as possible from the culture to expand/maintain HSPCs for clinical application. We cultured CB-CD34+ cells or CD34+ CD38- CD45RA- CD90+ CD49f+ (here we called CD34+ CD49f+) cells as HSPCs in serum-free medium supplemented with recombinant human serum albumin (rHSA) and cytokines for 7days. In case of CD34+ cells, CD34+ cells were maintained in the culture although more than half of cells were differentiated hematopoietic cells. As same as CD34+ cells, CD34+ CD49f+ cells were remained in culture and weaved with differentiated cells. Transplantation study revealed that HSPCs cultured in rHSA-containing medium for 7 days could produce human hematopoietic cells in irradiated NOD-scid-IL2Rgc-/- (NOG) mice for a long period. These results suggest that HSPCs can be expanded and/or maintained in the culture condition consisting of defined factors alone, although further analyses are required to clarify the absolute number and the potential of individual HSPCs after cultivation.
3182 - ROLE OF SPRED1 IN THE REGULATION OF SELF-RENEWAL AND AGING OF HEMATOPOIETIC STEM CELLS Yuko Tadokoro1, Akihiko Yoshimura2, Hiromitsu Nakauchi3, and Atsushi Hirao1 1 Cancer Research institute, Kanazawa University, Kanazawa, Ishikawa, Japan; 2Keio University School of Medicine, Shinjyuku-ku, Tokyo, Japan; 3The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Hematopoietic stem cell (HSC) self-renewal and differentiation are regulated by microenvironments. Although it has been demonstrated that stem cell factor (SCF)/c-Kit receptor signal plays an important role in the fate determination of HSCs, the molecular mechanisms of fine-tuning of the c-Kit signal have remained elusive. We show that Spred1, a negative regulator of SCF/c-Kit signaling, controls self-renewal activity of HSCs. Spred1 mRNA highly expressed in undifferentiated hematopoietic cells and its protein level was exclusively high in short-term HSCs/progenitor cells. Whereas overexpression of Spred1 in HSCs inhibited the hematopoietic reconstitution, Spred1-deficient HSCs showed the increased hematopoietic reconstitution capacity, indicating that Spred1 negatively controls HSC function in vivo. Spred1 deficiency increased colony-forming activity of HSCs in cell-cell contact dependent and c-Kit dependent manners in vitro. In addition, Spred1 deficiency enhanced ROCK-mediated cell spreading of HSCs, in response to SCF stimulation. Interestingly, ROCK inhibitor treatment normalized the reconstitution activity in vivo and colony forming activity in vitro, indicating that Spred1 deficiency caused enhancement of HSC function by promotion of ROCK-dependent actin polymerization. Expression level of Spred1 protein in HSCs of aged mice (O100 weeks old) was significantly higher than that of young mice (12-16 weeks old), suggesting that increased Spred1 causes aging phenotypes of HSCs. In serial transplantation experiments, normal HSCs lost repopulating capacity in the tertiary transplantation. Spred1 deficiency HSCs still maintained its activity, without any symptoms of leukemogenesis. These data indicate that Spred1 controls life span of hematopoietic cells, mediated by regulation of self-renewal of HSCs. Further studies of Spred1-mediated control of self-renewal activity of HSCs would lead to deep understanding of HSC aging.
3183 - DECLINED PRESENTATION THE EFFECTS OF INTERFERON a-2B ON DEFECTIVE BONE MARROW MESENCHYMAL STEM CELLS IN PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA Tiantian Sun, Cuicui Lv, Donglei Zhang, Mingen Lv, Xian Zhang, Wenjie Liu, Rongfeng Fu, Huiyuan Li, Feng Xue, Xiaofan Liu, Renchi Yang, and Lei Zhang State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Disease, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Bone marrow-derived mesenchymal stem cells (BM-MSCs) are key components of the hematopoietic microenvironment that may be involved in the pathogenesis of myeloproliferative neoplasms, such as essential thrombocythemia (ET). Interferon a has anti-tumor effects, though its effects in these conditions are unclear. Therefore, we investigated the biological and functional characteristics of BM-MSCs and their responses to interferon a-2b in patients with ET. BM-MSCs from ET patients and normal controls shared similar morphology, immunophenotype, and adipogenic differentiation potentials. BM-MSCs from ET patients had a higher proliferation capacity and lower osteogenic differentiation capacity, lower apoptosis rate, and impaired support of hematopoiesis. Furthermore, in cells from patients with ET, interferon a-2b increased the apoptosis rate and enhanced the expansion of CD34+ hematopoietic progenitor cells, but impaired differentiation into osteoblasts and adipocytes, as well as the ability to produce hematopoietic colony-forming unit megakaryocytes. In summary, abnormal BM-MSCs may contribute to the development of ET. Interferon a-2b may help restore normal function in bone marrow-derived mesenchymal stem cells from these patients.
3184 - ION TRANSPORT-ASSOCIATED MOLECULES AS THERAPEUTIC TARGETS AGAINST GLIOBLASTOMA STEM CELLS Tetsuya Takada, Kazuyuki Takata, and Eishi Ashihara Kyoto Pharmaceutical University, Kyoto, Japan Introduction: Recent evidences suggest that a minor subset of cancer cells, so-called cancer stem cells (CSCs), have renewal potential to develop tumors. Therefore, CSCs characterization is an important issue for developing therapeutic strategies against cancers. Cancer cells produce ATP through glycolysis even under a normoxic condition (Warburg’s effect), and they generate excessive acidic substances. Cancer cells keep intracellular pH in weak alkaline by transporting protons across plasma membranes of cancer cells to maintain their functions. Consequently, cancer microenvironments become acidified. Glioblastoma (GBM), the most malignant form of glioma, is one of the most aggressive human cancers with a low 5 year survival rate and a high recurrence rate. We hypothesize that the ion transport-related molecules regulate the maintenance and the functions of CSCs. In the present study, we investigated the expression of ion transport-related molecules in CSCs of GBM cells (GSCs). Methods: We examined the expressions of Na+-H+ exchangers (NHEs), monocarboxylate transporters (MCTs), and carbonic anhydrases (CAs) in GSCs. GSCs were generated using a cancer sphere formation assay. We also established hypoxia-adapted (HA) GBM cells that can be survived and proliferate under a hypoxic condition (O2 1%), and examined their expressions in these HA GBM cells (nonCSC/HA cells). We also analyzed influence on the proliferation of GSCs, non-CSCs using inhibitors of targets whose expression were increased in GSCs. Results and discussion: Immunofluorescent examinations showed that stem cell markers were detected in cancer spheres and that cancer spheres were hypoxic. In quantitative RTPCR analyses, stem cell markers were elevated in GSCs, moreover CA12 and MCT1 mRNA transcripts were elevated in CSC spheres but not in non-CSC/HA cells. Moreover, the inhibitors of CA12 or MCT1 suppressed the proliferation of non-CSCs and GSCs. In conclusion, CA12 and MCT1 maintain GSCs and these molecules are promising targets against GSCs.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3180 - MAINTENANCE AND EXPANSION OF HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS IN THE PRESENCE OF DEFINED FACTORS ALONE Kazuhiro Sudo1, Satoshi Yamazaki2, Hiromitsu Nakauchi2, and Yukio Nakamura1 1 RIKEN BRC, Tsukuba, Ibaraki, Japan; 2University of Tokyo, Tokyo, Japan Human hematopoietic stem and progenitor cells (HSPCs) in bone marrow (BM) or umbilical cord blood (CB) are very rare cell populations. Especially the number of HSPCs in CB derived from one donor is quite limited. Ex vivo expansion is thought to be a promising procedure to produce the sufficient number of human HSPC for clinical transplantations. Studies about ex vivo expansion of HSPCs have been reported. In many cases, animal-derived components, such as fetal bovine serum (FBS) and bovine serum albumin fraction V (BSA-V), are added to the culture. It is well known that different lot of BSA-V give different effects to HSPC proliferation. Most recently, we found that mouse hematopoietic stem cells (HSCs) cultivated in different lot of BSA-V-contained culture condition have different ability to reconstitute irradiated mouse hematopoiesis even if all BSA-V give the comparable level of cell proliferation level. This result strongly suggests that addition of animalderived component(s) to HSC-culture provoke the instability. In addition to the instability of experimental data, animal-derived components should be excluded as much as possible from the culture to expand/maintain HSPCs for clinical application. We cultured CB-CD34+ cells or CD34+ CD38- CD45RA- CD90+ CD49f+ (here we called CD34+ CD49f+) cells as HSPCs in serum-free medium supplemented with recombinant human serum albumin (rHSA) and cytokines for 7days. In case of CD34+ cells, CD34+ cells were maintained in the culture although more than half of cells were differentiated hematopoietic cells. As same as CD34+ cells, CD34+ CD49f+ cells were remained in culture and weaved with differentiated cells. Transplantation study revealed that HSPCs cultured in rHSA-containing medium for 7 days could produce human hematopoietic cells in irradiated NOD-scid-IL2Rgc-/- (NOG) mice for a long period. These results suggest that HSPCs can be expanded and/or maintained in the culture condition consisting of defined factors alone, although further analyses are required to clarify the absolute number and the potential of individual HSPCs after cultivation.
3182 - ROLE OF SPRED1 IN THE REGULATION OF SELF-RENEWAL AND AGING OF HEMATOPOIETIC STEM CELLS Yuko Tadokoro1, Akihiko Yoshimura2, Hiromitsu Nakauchi3, and Atsushi Hirao1 1 Cancer Research institute, Kanazawa University, Kanazawa, Ishikawa, Japan; 2Keio University School of Medicine, Shinjyuku-ku, Tokyo, Japan; 3The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan Hematopoietic stem cell (HSC) self-renewal and differentiation are regulated by microenvironments. Although it has been demonstrated that stem cell factor (SCF)/c-Kit receptor signal plays an important role in the fate determination of HSCs, the molecular mechanisms of fine-tuning of the c-Kit signal have remained elusive. We show that Spred1, a negative regulator of SCF/c-Kit signaling, controls self-renewal activity of HSCs. Spred1 mRNA highly expressed in undifferentiated hematopoietic cells and its protein level was exclusively high in short-term HSCs/progenitor cells. Whereas overexpression of Spred1 in HSCs inhibited the hematopoietic reconstitution, Spred1-deficient HSCs showed the increased hematopoietic reconstitution capacity, indicating that Spred1 negatively controls HSC function in vivo. Spred1 deficiency increased colony-forming activity of HSCs in cell-cell contact dependent and c-Kit dependent manners in vitro. In addition, Spred1 deficiency enhanced ROCK-mediated cell spreading of HSCs, in response to SCF stimulation. Interestingly, ROCK inhibitor treatment normalized the reconstitution activity in vivo and colony forming activity in vitro, indicating that Spred1 deficiency caused enhancement of HSC function by promotion of ROCK-dependent actin polymerization. Expression level of Spred1 protein in HSCs of aged mice (O100 weeks old) was significantly higher than that of young mice (12-16 weeks old), suggesting that increased Spred1 causes aging phenotypes of HSCs. In serial transplantation experiments, normal HSCs lost repopulating capacity in the tertiary transplantation. Spred1 deficiency HSCs still maintained its activity, without any symptoms of leukemogenesis. These data indicate that Spred1 controls life span of hematopoietic cells, mediated by regulation of self-renewal of HSCs. Further studies of Spred1-mediated control of self-renewal activity of HSCs would lead to deep understanding of HSC aging.
3183 - DECLINED PRESENTATION THE EFFECTS OF INTERFERON a-2B ON DEFECTIVE BONE MARROW MESENCHYMAL STEM CELLS IN PATIENTS WITH ESSENTIAL THROMBOCYTHEMIA Tiantian Sun, Cuicui Lv, Donglei Zhang, Mingen Lv, Xian Zhang, Wenjie Liu, Rongfeng Fu, Huiyuan Li, Feng Xue, Xiaofan Liu, Renchi Yang, and Lei Zhang State Key Laboratory of Experimental Hematology, Institute of Hematology and Hospital of Blood Disease, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China Bone marrow-derived mesenchymal stem cells (BM-MSCs) are key components of the hematopoietic microenvironment that may be involved in the pathogenesis of myeloproliferative neoplasms, such as essential thrombocythemia (ET). Interferon a has anti-tumor effects, though its effects in these conditions are unclear. Therefore, we investigated the biological and functional characteristics of BM-MSCs and their responses to interferon a-2b in patients with ET. BM-MSCs from ET patients and normal controls shared similar morphology, immunophenotype, and adipogenic differentiation potentials. BM-MSCs from ET patients had a higher proliferation capacity and lower osteogenic differentiation capacity, lower apoptosis rate, and impaired support of hematopoiesis. Furthermore, in cells from patients with ET, interferon a-2b increased the apoptosis rate and enhanced the expansion of CD34+ hematopoietic progenitor cells, but impaired differentiation into osteoblasts and adipocytes, as well as the ability to produce hematopoietic colony-forming unit megakaryocytes. In summary, abnormal BM-MSCs may contribute to the development of ET. Interferon a-2b may help restore normal function in bone marrow-derived mesenchymal stem cells from these patients.
3184 - ION TRANSPORT-ASSOCIATED MOLECULES AS THERAPEUTIC TARGETS AGAINST GLIOBLASTOMA STEM CELLS Tetsuya Takada, Kazuyuki Takata, and Eishi Ashihara Kyoto Pharmaceutical University, Kyoto, Japan Introduction: Recent evidences suggest that a minor subset of cancer cells, so-called cancer stem cells (CSCs), have renewal potential to develop tumors. Therefore, CSCs characterization is an important issue for developing therapeutic strategies against cancers. Cancer cells produce ATP through glycolysis even under a normoxic condition (Warburg’s effect), and they generate excessive acidic substances. Cancer cells keep intracellular pH in weak alkaline by transporting protons across plasma membranes of cancer cells to maintain their functions. Consequently, cancer microenvironments become acidified. Glioblastoma (GBM), the most malignant form of glioma, is one of the most aggressive human cancers with a low 5 year survival rate and a high recurrence rate. We hypothesize that the ion transport-related molecules regulate the maintenance and the functions of CSCs. In the present study, we investigated the expression of ion transport-related molecules in CSCs of GBM cells (GSCs). Methods: We examined the expressions of Na+-H+ exchangers (NHEs), monocarboxylate transporters (MCTs), and carbonic anhydrases (CAs) in GSCs. GSCs were generated using a cancer sphere formation assay. We also established hypoxia-adapted (HA) GBM cells that can be survived and proliferate under a hypoxic condition (O2 1%), and examined their expressions in these HA GBM cells (nonCSC/HA cells). We also analyzed influence on the proliferation of GSCs, non-CSCs using inhibitors of targets whose expression were increased in GSCs. Results and discussion: Immunofluorescent examinations showed that stem cell markers were detected in cancer spheres and that cancer spheres were hypoxic. In quantitative RTPCR analyses, stem cell markers were elevated in GSCs, moreover CA12 and MCT1 mRNA transcripts were elevated in CSC spheres but not in non-CSC/HA cells. Moreover, the inhibitors of CA12 or MCT1 suppressed the proliferation of non-CSCs and GSCs. In conclusion, CA12 and MCT1 maintain GSCs and these molecules are promising targets against GSCs.