NBEA: A NOVEL REGULATOR OF HEMATOPOIETIC STEM CELL IN VIVO REPOPULATION
(Abstract release date: 05/19/16)
EHA Library. Ganuza Fernandez M. 06/10/16; 135156; S123
Dr. Miguel Ganuza Fernandez
Contributions
Contributions
Abstract
Abstract: S123
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Hematopoietic Stem Cells (HSCs) differentiate to generate all blood cells in a hierarchical manner. This has been exploited in HSC transplantation (HSCT) to treat hematologic diseases. Better understanding the molecular mechanisms implicated in HSCT should improve current transplant protocols. In our recently published work (Holmfeldt et al. J.Exp.Med. 2016), we have identified 17 new regulators of HSCT. Neurobeachin (Nbea) scored as a positive regulator of HSCT. NBEA protein is localized near the Golgi apparatus and regulates vesicular and protein trafficking. Patients with a disrupted NBEA allele present platelets with an abnormal morphology.
Aims
To understand the role of Nbea in HSCT and normal hematopoiesis.
Methods
Nbea-/- mice. Bone marrow (BM) transplant: Competitive congenic transplant into lethally irradiated recipients: Test cells (CD45.2+), competitor cells (CD45.1+), recipient mice (CD45.1+/CD45.2+). Flow Cytometry Analysis of Peripheral Blood (PB) and BM. RNA-microarray.
Results
Both adult BM Lineage-Sca-1+c-Kit+ (LSK) and E14.5 Fetal Liver (FL) LSK cells lentivirally transduced with shRNAs targeting Nbea (>70% transcript knockdown), and competitively transplanted within 40 hours post-infection into lethally irradiated recipient mice, suffer from a strong defect in short- and long-term repopulating activity, as measured by PB chimerism of recipient mice (> 80% reduced). Thus, Nbea is required in LSK cells at different developmental stages for efficient HSCT. As transplanted Nbea-deficient cells are detected in the BM of recipients at 12 days post-transplantation, thus, a defect in homing or in LSK cell BM retention in recipients can be excluded. 4 months post-transplant, analysis of the major BM hematopoietic progenitor compartments (HPCs) of recipients showed a 50% decreased in the chimerism of Nbea-deficient cells in the HSC compartment. In contrast, Nbea-deficient cells were nearly absent from downstream progenitor compartments, suggesting a differentiation block.Nbea-/- mice die perinatally due to lack of synaptic transmissions There was no difference in the frequency of HPCs in the E14.5 FL of Nbea-/- and Nbea+/+ littermates. Nbea-/- E14.5 FL-LSK cells showed no defect in engraftment ability compared to Nbea+/+ controls, suggesting that embryonic plasticity has allowed for compensation. When CD45.2+ LSK cells derived from primary recipients of Nbea-/-E14.5 FL-LSK (CD45.2+) cells were isolated and transplanted into secondary recipients, a 25% reduction in repopulating activity was observed, supporting a role for Nbea in HSCT and HSC self-renewalTo understand how Nbea regulates HSC biology, we looked for differentially expressed genes using arrays on total RNA isolated from E14.5 FL-CD150+CD48- LSK cells sorted from both Nbea+/+ and Nbea-/- embryos. Gene sets implicated in Snare interactions, vesicular transport, and Adherens junctions interactions were upregulated in Nbea-/- cells, supporting a role for Nbea in these processes.
Conclusion
Nbea is a bona fide regulator of HSC in vivo repopulation. Although Nbea is dispensable for native hematopoiesis, differences present in genes with a role in both vesicular trafficking and Adherens Junctions Interactions highlight the role of Nbea in these processes and how important they are for proper HSC interactions with the BM niche during HSCT.
Session topic: Stem cell transplantation - Experimental
Keyword(s): Bone marrow transplant, Hematopoietic stem and progenitor cells
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Hematopoietic Stem Cells (HSCs) differentiate to generate all blood cells in a hierarchical manner. This has been exploited in HSC transplantation (HSCT) to treat hematologic diseases. Better understanding the molecular mechanisms implicated in HSCT should improve current transplant protocols. In our recently published work (Holmfeldt et al. J.Exp.Med. 2016), we have identified 17 new regulators of HSCT. Neurobeachin (Nbea) scored as a positive regulator of HSCT. NBEA protein is localized near the Golgi apparatus and regulates vesicular and protein trafficking. Patients with a disrupted NBEA allele present platelets with an abnormal morphology.
Aims
To understand the role of Nbea in HSCT and normal hematopoiesis.
Methods
Nbea-/- mice. Bone marrow (BM) transplant: Competitive congenic transplant into lethally irradiated recipients: Test cells (CD45.2+), competitor cells (CD45.1+), recipient mice (CD45.1+/CD45.2+). Flow Cytometry Analysis of Peripheral Blood (PB) and BM. RNA-microarray.
Results
Both adult BM Lineage-Sca-1+c-Kit+ (LSK) and E14.5 Fetal Liver (FL) LSK cells lentivirally transduced with shRNAs targeting Nbea (>70% transcript knockdown), and competitively transplanted within 40 hours post-infection into lethally irradiated recipient mice, suffer from a strong defect in short- and long-term repopulating activity, as measured by PB chimerism of recipient mice (> 80% reduced). Thus, Nbea is required in LSK cells at different developmental stages for efficient HSCT. As transplanted Nbea-deficient cells are detected in the BM of recipients at 12 days post-transplantation, thus, a defect in homing or in LSK cell BM retention in recipients can be excluded. 4 months post-transplant, analysis of the major BM hematopoietic progenitor compartments (HPCs) of recipients showed a 50% decreased in the chimerism of Nbea-deficient cells in the HSC compartment. In contrast, Nbea-deficient cells were nearly absent from downstream progenitor compartments, suggesting a differentiation block.Nbea-/- mice die perinatally due to lack of synaptic transmissions There was no difference in the frequency of HPCs in the E14.5 FL of Nbea-/- and Nbea+/+ littermates. Nbea-/- E14.5 FL-LSK cells showed no defect in engraftment ability compared to Nbea+/+ controls, suggesting that embryonic plasticity has allowed for compensation. When CD45.2+ LSK cells derived from primary recipients of Nbea-/-E14.5 FL-LSK (CD45.2+) cells were isolated and transplanted into secondary recipients, a 25% reduction in repopulating activity was observed, supporting a role for Nbea in HSCT and HSC self-renewalTo understand how Nbea regulates HSC biology, we looked for differentially expressed genes using arrays on total RNA isolated from E14.5 FL-CD150+CD48- LSK cells sorted from both Nbea+/+ and Nbea-/- embryos. Gene sets implicated in Snare interactions, vesicular transport, and Adherens junctions interactions were upregulated in Nbea-/- cells, supporting a role for Nbea in these processes.
Conclusion
Nbea is a bona fide regulator of HSC in vivo repopulation. Although Nbea is dispensable for native hematopoiesis, differences present in genes with a role in both vesicular trafficking and Adherens Junctions Interactions highlight the role of Nbea in these processes and how important they are for proper HSC interactions with the BM niche during HSCT.
Session topic: Stem cell transplantation - Experimental
Keyword(s): Bone marrow transplant, Hematopoietic stem and progenitor cells
Abstract: S123
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Hematopoietic Stem Cells (HSCs) differentiate to generate all blood cells in a hierarchical manner. This has been exploited in HSC transplantation (HSCT) to treat hematologic diseases. Better understanding the molecular mechanisms implicated in HSCT should improve current transplant protocols. In our recently published work (Holmfeldt et al. J.Exp.Med. 2016), we have identified 17 new regulators of HSCT. Neurobeachin (Nbea) scored as a positive regulator of HSCT. NBEA protein is localized near the Golgi apparatus and regulates vesicular and protein trafficking. Patients with a disrupted NBEA allele present platelets with an abnormal morphology.
Aims
To understand the role of Nbea in HSCT and normal hematopoiesis.
Methods
Nbea-/- mice. Bone marrow (BM) transplant: Competitive congenic transplant into lethally irradiated recipients: Test cells (CD45.2+), competitor cells (CD45.1+), recipient mice (CD45.1+/CD45.2+). Flow Cytometry Analysis of Peripheral Blood (PB) and BM. RNA-microarray.
Results
Both adult BM Lineage-Sca-1+c-Kit+ (LSK) and E14.5 Fetal Liver (FL) LSK cells lentivirally transduced with shRNAs targeting Nbea (>70% transcript knockdown), and competitively transplanted within 40 hours post-infection into lethally irradiated recipient mice, suffer from a strong defect in short- and long-term repopulating activity, as measured by PB chimerism of recipient mice (> 80% reduced). Thus, Nbea is required in LSK cells at different developmental stages for efficient HSCT. As transplanted Nbea-deficient cells are detected in the BM of recipients at 12 days post-transplantation, thus, a defect in homing or in LSK cell BM retention in recipients can be excluded. 4 months post-transplant, analysis of the major BM hematopoietic progenitor compartments (HPCs) of recipients showed a 50% decreased in the chimerism of Nbea-deficient cells in the HSC compartment. In contrast, Nbea-deficient cells were nearly absent from downstream progenitor compartments, suggesting a differentiation block.Nbea-/- mice die perinatally due to lack of synaptic transmissions There was no difference in the frequency of HPCs in the E14.5 FL of Nbea-/- and Nbea+/+ littermates. Nbea-/- E14.5 FL-LSK cells showed no defect in engraftment ability compared to Nbea+/+ controls, suggesting that embryonic plasticity has allowed for compensation. When CD45.2+ LSK cells derived from primary recipients of Nbea-/-E14.5 FL-LSK (CD45.2+) cells were isolated and transplanted into secondary recipients, a 25% reduction in repopulating activity was observed, supporting a role for Nbea in HSCT and HSC self-renewalTo understand how Nbea regulates HSC biology, we looked for differentially expressed genes using arrays on total RNA isolated from E14.5 FL-CD150+CD48- LSK cells sorted from both Nbea+/+ and Nbea-/- embryos. Gene sets implicated in Snare interactions, vesicular transport, and Adherens junctions interactions were upregulated in Nbea-/- cells, supporting a role for Nbea in these processes.
Conclusion
Nbea is a bona fide regulator of HSC in vivo repopulation. Although Nbea is dispensable for native hematopoiesis, differences present in genes with a role in both vesicular trafficking and Adherens Junctions Interactions highlight the role of Nbea in these processes and how important they are for proper HSC interactions with the BM niche during HSCT.
Session topic: Stem cell transplantation - Experimental
Keyword(s): Bone marrow transplant, Hematopoietic stem and progenitor cells
Type: Oral Presentation
Presentation during EHA21: On Friday, June 10, 2016 from 11:30 - 11:45
Location: Hall C13
Background
Hematopoietic Stem Cells (HSCs) differentiate to generate all blood cells in a hierarchical manner. This has been exploited in HSC transplantation (HSCT) to treat hematologic diseases. Better understanding the molecular mechanisms implicated in HSCT should improve current transplant protocols. In our recently published work (Holmfeldt et al. J.Exp.Med. 2016), we have identified 17 new regulators of HSCT. Neurobeachin (Nbea) scored as a positive regulator of HSCT. NBEA protein is localized near the Golgi apparatus and regulates vesicular and protein trafficking. Patients with a disrupted NBEA allele present platelets with an abnormal morphology.
Aims
To understand the role of Nbea in HSCT and normal hematopoiesis.
Methods
Nbea-/- mice. Bone marrow (BM) transplant: Competitive congenic transplant into lethally irradiated recipients: Test cells (CD45.2+), competitor cells (CD45.1+), recipient mice (CD45.1+/CD45.2+). Flow Cytometry Analysis of Peripheral Blood (PB) and BM. RNA-microarray.
Results
Both adult BM Lineage-Sca-1+c-Kit+ (LSK) and E14.5 Fetal Liver (FL) LSK cells lentivirally transduced with shRNAs targeting Nbea (>70% transcript knockdown), and competitively transplanted within 40 hours post-infection into lethally irradiated recipient mice, suffer from a strong defect in short- and long-term repopulating activity, as measured by PB chimerism of recipient mice (> 80% reduced). Thus, Nbea is required in LSK cells at different developmental stages for efficient HSCT. As transplanted Nbea-deficient cells are detected in the BM of recipients at 12 days post-transplantation, thus, a defect in homing or in LSK cell BM retention in recipients can be excluded. 4 months post-transplant, analysis of the major BM hematopoietic progenitor compartments (HPCs) of recipients showed a 50% decreased in the chimerism of Nbea-deficient cells in the HSC compartment. In contrast, Nbea-deficient cells were nearly absent from downstream progenitor compartments, suggesting a differentiation block.Nbea-/- mice die perinatally due to lack of synaptic transmissions There was no difference in the frequency of HPCs in the E14.5 FL of Nbea-/- and Nbea+/+ littermates. Nbea-/- E14.5 FL-LSK cells showed no defect in engraftment ability compared to Nbea+/+ controls, suggesting that embryonic plasticity has allowed for compensation. When CD45.2+ LSK cells derived from primary recipients of Nbea-/-E14.5 FL-LSK (CD45.2+) cells were isolated and transplanted into secondary recipients, a 25% reduction in repopulating activity was observed, supporting a role for Nbea in HSCT and HSC self-renewalTo understand how Nbea regulates HSC biology, we looked for differentially expressed genes using arrays on total RNA isolated from E14.5 FL-CD150+CD48- LSK cells sorted from both Nbea+/+ and Nbea-/- embryos. Gene sets implicated in Snare interactions, vesicular transport, and Adherens junctions interactions were upregulated in Nbea-/- cells, supporting a role for Nbea in these processes.
Conclusion
Nbea is a bona fide regulator of HSC in vivo repopulation. Although Nbea is dispensable for native hematopoiesis, differences present in genes with a role in both vesicular trafficking and Adherens Junctions Interactions highlight the role of Nbea in these processes and how important they are for proper HSC interactions with the BM niche during HSCT.
Session topic: Stem cell transplantation - Experimental
Keyword(s): Bone marrow transplant, Hematopoietic stem and progenitor cells
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