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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

Gene expression analysis of LSK cells from dnMpl mice.(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [13,15,34,35,46,47,54] or based on the KEGG database.
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pone.0131866.g005: Gene expression analysis of LSK cells from dnMpl mice.(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [13,15,34,35,46,47,54] or based on the KEGG database.

Mentions: To further elucidate the molecular effects of dnMpl expression on HSPC, we performed microarray analysis on LSK cells from dnMpl and GFP control mice sorted eight weeks after transplantation (Fig 5A). In the transplanted mice approximately 50% of the LSK cells were dnMpl positive. For comparison of dnMpl expressing versus non expressing cells we sorted dnMpl positive and negative LSK cells from the same mice (Fig 5B). We determined which genes were differentially expressed between dnMpl+ and ctrl LSK cells. Strikingly, hierarchical clustering and PCA analysis on these genes revealed that gene expression in dnMpl+ and dnMpl- LSK cells in mice transplanted with dnMpl-cells was very similar, as they clustered together, and were clearly distinct from the control mice (Fig 5C and 5D). This indicated that expression of dnMpl in the bone marrow impacted transgene positive and negative LSK-cells in a similar way.


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)

Gene expression analysis of LSK cells from dnMpl mice.(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [13,15,34,35,46,47,54] or based on the KEGG database.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493002&req=5

pone.0131866.g005: Gene expression analysis of LSK cells from dnMpl mice.(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [13,15,34,35,46,47,54] or based on the KEGG database.
Mentions: To further elucidate the molecular effects of dnMpl expression on HSPC, we performed microarray analysis on LSK cells from dnMpl and GFP control mice sorted eight weeks after transplantation (Fig 5A). In the transplanted mice approximately 50% of the LSK cells were dnMpl positive. For comparison of dnMpl expressing versus non expressing cells we sorted dnMpl positive and negative LSK cells from the same mice (Fig 5B). We determined which genes were differentially expressed between dnMpl+ and ctrl LSK cells. Strikingly, hierarchical clustering and PCA analysis on these genes revealed that gene expression in dnMpl+ and dnMpl- LSK cells in mice transplanted with dnMpl-cells was very similar, as they clustered together, and were clearly distinct from the control mice (Fig 5C and 5D). This indicated that expression of dnMpl in the bone marrow impacted transgene positive and negative LSK-cells in a similar way.

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