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Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Kohlscheen S, Wintterle S, Schwarzer A, Kamp C, Brugman MH, Breuer DC, Büsche G, Baum C, Modlich U - PLoS ONE (2015)

Bottom Line: Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding.The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population).We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations.

View Article: PubMed Central - PubMed

Affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.

ABSTRACT
Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

No MeSH data available.


Related in: MedlinePlus

dnMpl expression in vivo causes thrombocytopenia.(A) Blood cell counts of transplanted mice six and 12–14 weeks after transplantation. Platelet counts remained significantly reduced in the dnMpl group, whereas, the hematocrit was unchanged in both groups. Slightly reduced WBC counts were observed at late time points after transplantation in the dnMpl group. (*** p<0.005, Students t-test) HCT: hematocrit, WBC: white blood cells. (B) Correlation of dnMpl expression on leukocytes with platelet counts. Blood of mice transplanted with dnMpl or GFP control transduced BM cells was taken at different time points after transplantation, platelet counts and transgene expression were measured by automated blood cell counts or flow cytometry, respectively. dnMpl expression results in decreased platelet counts. With declining dnMpl expression the platelet counts adjust to physiological levels. (C) Thpo plasma levels were determined in the blood of transplanted mice by ELISA, 12 weeks after transplantation or untransplanted wildtype and Mpl-/- mice. (*p<0.05, **p<0.01, Students t-test). (D) Receptor uptake in 32D cells. 32D cells were transduced with either wtMpl or dnMpl expressing vectors and stimulated with 50ng/mL mThpo or not. Cells were then fixed at different time points after Thpo has been added and surface expression of either receptor was measured by flow cytometry using an anti-HA Biotin conjugated primary and Streptavidin-PE secondary antibody.
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pone.0131866.g002: dnMpl expression in vivo causes thrombocytopenia.(A) Blood cell counts of transplanted mice six and 12–14 weeks after transplantation. Platelet counts remained significantly reduced in the dnMpl group, whereas, the hematocrit was unchanged in both groups. Slightly reduced WBC counts were observed at late time points after transplantation in the dnMpl group. (*** p<0.005, Students t-test) HCT: hematocrit, WBC: white blood cells. (B) Correlation of dnMpl expression on leukocytes with platelet counts. Blood of mice transplanted with dnMpl or GFP control transduced BM cells was taken at different time points after transplantation, platelet counts and transgene expression were measured by automated blood cell counts or flow cytometry, respectively. dnMpl expression results in decreased platelet counts. With declining dnMpl expression the platelet counts adjust to physiological levels. (C) Thpo plasma levels were determined in the blood of transplanted mice by ELISA, 12 weeks after transplantation or untransplanted wildtype and Mpl-/- mice. (*p<0.05, **p<0.01, Students t-test). (D) Receptor uptake in 32D cells. 32D cells were transduced with either wtMpl or dnMpl expressing vectors and stimulated with 50ng/mL mThpo or not. Cells were then fixed at different time points after Thpo has been added and surface expression of either receptor was measured by flow cytometry using an anti-HA Biotin conjugated primary and Streptavidin-PE secondary antibody.

Mentions: In the experiments 1 and 2, we transplanted five and ten wt mice per group with BM cells expressing dnMpl, GFP or trCD34 as control (Table 1). Cell surface expression of dnMpl and trCD34 on peripheral blood leukocytes was confirmed by live cell staining and analysis of the HA-tag using flow cytometry (S1B Fig). dnMpl expression was detected on all hematopoietic lineages in the peripheral blood of transplanted mice by flow cytometric analysis (S2 Fig). Platelet counts were severely reduced in dnMpl mice six weeks after BMT (254+/-25 versus 1162+/-95 x 103/μl in control mice, p<0.0001) and remained low (Fig 2A) with the exception of five mice with low levels of dnMpl expression. We found that ~6% of dnMpl positive cells in the blood (based on the expression in leukocytes) were sufficient to induce thrombocytopenia (Fig 2B). The hematocrit was unaffected by the expression of dnMpl, but 12 to 14 weeks after BMT, dnMpl mice showed a tendency of reduced white blood cell (WBC) counts (5.9+/- 0.86 x103/μl versus 8.34+/-1.11 x103/μl in control mice, p = 0.097). However, the expression of dnMpl did not affect the hematopoietic contribution of the different leukocyte lineages in the blood (S3 Fig). Thus, dnMpl expression had no effect on hematopoietic differentiation of non-thrombocytic lineages.


Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Kohlscheen S, Wintterle S, Schwarzer A, Kamp C, Brugman MH, Breuer DC, Büsche G, Baum C, Modlich U - PLoS ONE (2015)

dnMpl expression in vivo causes thrombocytopenia.(A) Blood cell counts of transplanted mice six and 12–14 weeks after transplantation. Platelet counts remained significantly reduced in the dnMpl group, whereas, the hematocrit was unchanged in both groups. Slightly reduced WBC counts were observed at late time points after transplantation in the dnMpl group. (*** p<0.005, Students t-test) HCT: hematocrit, WBC: white blood cells. (B) Correlation of dnMpl expression on leukocytes with platelet counts. Blood of mice transplanted with dnMpl or GFP control transduced BM cells was taken at different time points after transplantation, platelet counts and transgene expression were measured by automated blood cell counts or flow cytometry, respectively. dnMpl expression results in decreased platelet counts. With declining dnMpl expression the platelet counts adjust to physiological levels. (C) Thpo plasma levels were determined in the blood of transplanted mice by ELISA, 12 weeks after transplantation or untransplanted wildtype and Mpl-/- mice. (*p<0.05, **p<0.01, Students t-test). (D) Receptor uptake in 32D cells. 32D cells were transduced with either wtMpl or dnMpl expressing vectors and stimulated with 50ng/mL mThpo or not. Cells were then fixed at different time points after Thpo has been added and surface expression of either receptor was measured by flow cytometry using an anti-HA Biotin conjugated primary and Streptavidin-PE secondary antibody.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493002&req=5

pone.0131866.g002: dnMpl expression in vivo causes thrombocytopenia.(A) Blood cell counts of transplanted mice six and 12–14 weeks after transplantation. Platelet counts remained significantly reduced in the dnMpl group, whereas, the hematocrit was unchanged in both groups. Slightly reduced WBC counts were observed at late time points after transplantation in the dnMpl group. (*** p<0.005, Students t-test) HCT: hematocrit, WBC: white blood cells. (B) Correlation of dnMpl expression on leukocytes with platelet counts. Blood of mice transplanted with dnMpl or GFP control transduced BM cells was taken at different time points after transplantation, platelet counts and transgene expression were measured by automated blood cell counts or flow cytometry, respectively. dnMpl expression results in decreased platelet counts. With declining dnMpl expression the platelet counts adjust to physiological levels. (C) Thpo plasma levels were determined in the blood of transplanted mice by ELISA, 12 weeks after transplantation or untransplanted wildtype and Mpl-/- mice. (*p<0.05, **p<0.01, Students t-test). (D) Receptor uptake in 32D cells. 32D cells were transduced with either wtMpl or dnMpl expressing vectors and stimulated with 50ng/mL mThpo or not. Cells were then fixed at different time points after Thpo has been added and surface expression of either receptor was measured by flow cytometry using an anti-HA Biotin conjugated primary and Streptavidin-PE secondary antibody.
Mentions: In the experiments 1 and 2, we transplanted five and ten wt mice per group with BM cells expressing dnMpl, GFP or trCD34 as control (Table 1). Cell surface expression of dnMpl and trCD34 on peripheral blood leukocytes was confirmed by live cell staining and analysis of the HA-tag using flow cytometry (S1B Fig). dnMpl expression was detected on all hematopoietic lineages in the peripheral blood of transplanted mice by flow cytometric analysis (S2 Fig). Platelet counts were severely reduced in dnMpl mice six weeks after BMT (254+/-25 versus 1162+/-95 x 103/μl in control mice, p<0.0001) and remained low (Fig 2A) with the exception of five mice with low levels of dnMpl expression. We found that ~6% of dnMpl positive cells in the blood (based on the expression in leukocytes) were sufficient to induce thrombocytopenia (Fig 2B). The hematocrit was unaffected by the expression of dnMpl, but 12 to 14 weeks after BMT, dnMpl mice showed a tendency of reduced white blood cell (WBC) counts (5.9+/- 0.86 x103/μl versus 8.34+/-1.11 x103/μl in control mice, p = 0.097). However, the expression of dnMpl did not affect the hematopoietic contribution of the different leukocyte lineages in the blood (S3 Fig). Thus, dnMpl expression had no effect on hematopoietic differentiation of non-thrombocytic lineages.

Bottom Line: Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding.The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population).We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations.

View Article: PubMed Central - PubMed

Affiliation: Research Group for Gene Modification in Stem Cells, LOEWE Center for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany; Institute of Experimental Hematology; Hannover Medical School, Hannover, Germany.

ABSTRACT
Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

No MeSH data available.


Related in: MedlinePlus