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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

Cell cycle analysis and cell surface molecule expression on LSK cells in dnMpl mice.(A) Venn diagram showing the overlap of the leading edge genes from the Gene Set Enrichment Analysis comparing our dataset with the data of [15,34,54]. (B) Expression of essential players in the cell cycle control. Displayed are the log2(fold-change) values determined based on the microarray analysis in comparison to control. (C) Cell cycle analysis of LSK cells of dnMpl and GFP control transplanted mice. Total BM cells were stained and gated for the LSK or LSK, CD34- cell population. Cell cycle status was determined by staining with Ki-67 and Hoechst 33342. A representative example of the flow cytometric analysis is shown (left picture) and the results summarized (right graph). Significantly less LSK and LSK, CD34- cells are within the G0 phase of the cell cycle (*p<0.05, n = 3). (D) Wild-type mice were transplanted with dnMpl or truncated (tr)CD34 (as control) expressing BM cells. At 16 weeks post transplantation total BM cells were stained for LSK cells, each BM sample was then splitted into three subsamples to stain for the different surface molecules: TIE2, EPCR (CD201), ESAM1. As controls, cell surface expression was also detected in untransplanted wt, Mpl-/- and Thpo-/- mice. The difference between the percentages of surface marker positive cells was significant, as indicated: *p<0.05, **p<0.01, ***p<0.005. (wt, Mpl-/-, Thpo-/-: n = 6; dnMpl, trCD34: n = 9).
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pone.0131866.g006: Cell cycle analysis and cell surface molecule expression on LSK cells in dnMpl mice.(A) Venn diagram showing the overlap of the leading edge genes from the Gene Set Enrichment Analysis comparing our dataset with the data of [15,34,54]. (B) Expression of essential players in the cell cycle control. Displayed are the log2(fold-change) values determined based on the microarray analysis in comparison to control. (C) Cell cycle analysis of LSK cells of dnMpl and GFP control transplanted mice. Total BM cells were stained and gated for the LSK or LSK, CD34- cell population. Cell cycle status was determined by staining with Ki-67 and Hoechst 33342. A representative example of the flow cytometric analysis is shown (left picture) and the results summarized (right graph). Significantly less LSK and LSK, CD34- cells are within the G0 phase of the cell cycle (*p<0.05, n = 3). (D) Wild-type mice were transplanted with dnMpl or truncated (tr)CD34 (as control) expressing BM cells. At 16 weeks post transplantation total BM cells were stained for LSK cells, each BM sample was then splitted into three subsamples to stain for the different surface molecules: TIE2, EPCR (CD201), ESAM1. As controls, cell surface expression was also detected in untransplanted wt, Mpl-/- and Thpo-/- mice. The difference between the percentages of surface marker positive cells was significant, as indicated: *p<0.05, **p<0.01, ***p<0.005. (wt, Mpl-/-, Thpo-/-: n = 6; dnMpl, trCD34: n = 9).

Mentions: We next sought to analyze to which cellular pathways and processes the affected genes belonged to. Gene set enrichment analysis (GSEA) of expression data from dnMpl+ and dnMpl- LSK cells of the same mice was performed separately and compared to LSK cells from control transplanted mice. In agreement with the hierarchical clustering and PC analyses similar gene set enrichment results were obtained with the dnMpl+ and dnMpl- expression profiles (Fig 5E, S8 Fig). Consistent with the Mpl-deficient phenotype, the two main downstream signaling pathways JAK-STAT and PI3K/AKT were downregulated. As another important signaling pathway for HSC we found that Wnt-signaling was downregulated in the dnMpl-mice. Most importantly, LSK cells from dnMpl-mice displayed a striking loss of hematopoietic stem-cell associated expression signatures, e.g. the signature of long term HSC [34] and human CD34+HSC [35], and the Thpo-responsive signature of HSC [15]. Gene expression analysis confirmed the induction of stem cell defects by dnMpl expression. This is in agreement with the role of Thpo/Mpl signaling in HSC maintenance and the loss of this signaling will inevitably cause HSC defects [6,33,36]. By comparing the leading edge genes of the aforementioned gene set enrichment analysis we detected substantial overlap of HSC-associated genes that are also deregulated in our dataset (e.g. Esam1, Plxdc2, Tie2, Prdm16, Procr/Epcr, MycN, Fhl1, HoxA5, Socs2;Fig 6A).


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)

Cell cycle analysis and cell surface molecule expression on LSK cells in dnMpl mice.(A) Venn diagram showing the overlap of the leading edge genes from the Gene Set Enrichment Analysis comparing our dataset with the data of [15,34,54]. (B) Expression of essential players in the cell cycle control. Displayed are the log2(fold-change) values determined based on the microarray analysis in comparison to control. (C) Cell cycle analysis of LSK cells of dnMpl and GFP control transplanted mice. Total BM cells were stained and gated for the LSK or LSK, CD34- cell population. Cell cycle status was determined by staining with Ki-67 and Hoechst 33342. A representative example of the flow cytometric analysis is shown (left picture) and the results summarized (right graph). Significantly less LSK and LSK, CD34- cells are within the G0 phase of the cell cycle (*p<0.05, n = 3). (D) Wild-type mice were transplanted with dnMpl or truncated (tr)CD34 (as control) expressing BM cells. At 16 weeks post transplantation total BM cells were stained for LSK cells, each BM sample was then splitted into three subsamples to stain for the different surface molecules: TIE2, EPCR (CD201), ESAM1. As controls, cell surface expression was also detected in untransplanted wt, Mpl-/- and Thpo-/- mice. The difference between the percentages of surface marker positive cells was significant, as indicated: *p<0.05, **p<0.01, ***p<0.005. (wt, Mpl-/-, Thpo-/-: n = 6; dnMpl, trCD34: n = 9).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493002&req=5

pone.0131866.g006: Cell cycle analysis and cell surface molecule expression on LSK cells in dnMpl mice.(A) Venn diagram showing the overlap of the leading edge genes from the Gene Set Enrichment Analysis comparing our dataset with the data of [15,34,54]. (B) Expression of essential players in the cell cycle control. Displayed are the log2(fold-change) values determined based on the microarray analysis in comparison to control. (C) Cell cycle analysis of LSK cells of dnMpl and GFP control transplanted mice. Total BM cells were stained and gated for the LSK or LSK, CD34- cell population. Cell cycle status was determined by staining with Ki-67 and Hoechst 33342. A representative example of the flow cytometric analysis is shown (left picture) and the results summarized (right graph). Significantly less LSK and LSK, CD34- cells are within the G0 phase of the cell cycle (*p<0.05, n = 3). (D) Wild-type mice were transplanted with dnMpl or truncated (tr)CD34 (as control) expressing BM cells. At 16 weeks post transplantation total BM cells were stained for LSK cells, each BM sample was then splitted into three subsamples to stain for the different surface molecules: TIE2, EPCR (CD201), ESAM1. As controls, cell surface expression was also detected in untransplanted wt, Mpl-/- and Thpo-/- mice. The difference between the percentages of surface marker positive cells was significant, as indicated: *p<0.05, **p<0.01, ***p<0.005. (wt, Mpl-/-, Thpo-/-: n = 6; dnMpl, trCD34: n = 9).
Mentions: We next sought to analyze to which cellular pathways and processes the affected genes belonged to. Gene set enrichment analysis (GSEA) of expression data from dnMpl+ and dnMpl- LSK cells of the same mice was performed separately and compared to LSK cells from control transplanted mice. In agreement with the hierarchical clustering and PC analyses similar gene set enrichment results were obtained with the dnMpl+ and dnMpl- expression profiles (Fig 5E, S8 Fig). Consistent with the Mpl-deficient phenotype, the two main downstream signaling pathways JAK-STAT and PI3K/AKT were downregulated. As another important signaling pathway for HSC we found that Wnt-signaling was downregulated in the dnMpl-mice. Most importantly, LSK cells from dnMpl-mice displayed a striking loss of hematopoietic stem-cell associated expression signatures, e.g. the signature of long term HSC [34] and human CD34+HSC [35], and the Thpo-responsive signature of HSC [15]. Gene expression analysis confirmed the induction of stem cell defects by dnMpl expression. This is in agreement with the role of Thpo/Mpl signaling in HSC maintenance and the loss of this signaling will inevitably cause HSC defects [6,33,36]. By comparing the leading edge genes of the aforementioned gene set enrichment analysis we detected substantial overlap of HSC-associated genes that are also deregulated in our dataset (e.g. Esam1, Plxdc2, Tie2, Prdm16, Procr/Epcr, MycN, Fhl1, HoxA5, Socs2;Fig 6A).

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