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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

Impact of dnMpl expression on the HSC compartment.(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, Mpl-/-, Thpo-/-). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to Mpl-/- mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.
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pone.0131866.g004: Impact of dnMpl expression on the HSC compartment.(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, Mpl-/-, Thpo-/-). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to Mpl-/- mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.

Mentions: The survival of primary recipients of dnMpl or control transduced BM was normal (dnMpl n = 25, control n = 20). After serial transplantation, however, secondary dnMpl recipients died due to graft failures starting from three weeks after transplantation while survival of secondary control mice was not impaired (experiment 3, dnMpl n = 10, control n = 6, Fig 4A).


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)

Impact of dnMpl expression on the HSC compartment.(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, Mpl-/-, Thpo-/-). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to Mpl-/- mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131866.g004: Impact of dnMpl expression on the HSC compartment.(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, Mpl-/-, Thpo-/-). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to Mpl-/- mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x107 CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.
Mentions: The survival of primary recipients of dnMpl or control transduced BM was normal (dnMpl n = 25, control n = 20). After serial transplantation, however, secondary dnMpl recipients died due to graft failures starting from three weeks after transplantation while survival of secondary control mice was not impaired (experiment 3, dnMpl n = 10, control n = 6, Fig 4A).

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