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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

Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.
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pone.0131866.g007: Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.

Mentions: Defects in Mpl signaling have been linked to familial aplastic anemia [42] and CAMT. Having established that inhibition of Mpl-signaling by dnMpl causes progressive BM-failure in mice we sought to analyze whether there was an overlap that would indicate functional impairment of Mpl signaling in human bone marrow failure syndromes. We therefore compared the gene expression profile of dnMpl LSK cells with expression profiles from CD34+ cells of patients with severe aplastic anemia (SAA) and refractory cytopenia (RC) published by Fischer and colleagues [43]. Human CD34+ cells are ~100-times less enriched for LT-HSC than murine LSK cells [44]. However, information of the gene expression in HSC of human aplastic anemia patients is difficult to obtain due to the low number of HSC in this disease. Also, in contrast to our mouse model, in humans SAA are mostly caused by autoimmune attack of CD34+ cells and gene expression analysis by Fisher and colleagues confirmed this (upregulated genes in immune and stress response and cell death) [43]. In our analysis we focused on gene sets associated with hematopoietic progenitors and stem cells. GSEA suggested that the signature in RC/SAA CD34+ cells was characterized by a severe loss of stem cell associated genes and an upregulation of genes that indicate lympho-myeloid differentiation, very similar to the effects seen upon expression of dnMpl (Fig 7A). Strikingly, MPL itself was one of the most downregulated genes in both SAA and RC with a mean log2FC of -3.5 (p<10−7, S11 Fig). In contrast to dnMpl LSK cells, cell cycle promoting genes were downregulated in CD34+ HSC from RC and SAA. By comparing gene deregulations based on scores which correlate the expression value to their respective controls in the murine dnMpl and human SAA/RC expression profiles, we identified genes with a similar positive and negative regulation (Fig 7A, S1–S4 Tables). Although there was no overall correlation between human and murine scores, important stem cell genes were similarly deregulated in both profiles.


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)

Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131866.g007: Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.
Mentions: Defects in Mpl signaling have been linked to familial aplastic anemia [42] and CAMT. Having established that inhibition of Mpl-signaling by dnMpl causes progressive BM-failure in mice we sought to analyze whether there was an overlap that would indicate functional impairment of Mpl signaling in human bone marrow failure syndromes. We therefore compared the gene expression profile of dnMpl LSK cells with expression profiles from CD34+ cells of patients with severe aplastic anemia (SAA) and refractory cytopenia (RC) published by Fischer and colleagues [43]. Human CD34+ cells are ~100-times less enriched for LT-HSC than murine LSK cells [44]. However, information of the gene expression in HSC of human aplastic anemia patients is difficult to obtain due to the low number of HSC in this disease. Also, in contrast to our mouse model, in humans SAA are mostly caused by autoimmune attack of CD34+ cells and gene expression analysis by Fisher and colleagues confirmed this (upregulated genes in immune and stress response and cell death) [43]. In our analysis we focused on gene sets associated with hematopoietic progenitors and stem cells. GSEA suggested that the signature in RC/SAA CD34+ cells was characterized by a severe loss of stem cell associated genes and an upregulation of genes that indicate lympho-myeloid differentiation, very similar to the effects seen upon expression of dnMpl (Fig 7A). Strikingly, MPL itself was one of the most downregulated genes in both SAA and RC with a mean log2FC of -3.5 (p<10−7, S11 Fig). In contrast to dnMpl LSK cells, cell cycle promoting genes were downregulated in CD34+ HSC from RC and SAA. By comparing gene deregulations based on scores which correlate the expression value to their respective controls in the murine dnMpl and human SAA/RC expression profiles, we identified genes with a similar positive and negative regulation (Fig 7A, S1–S4 Tables). Although there was no overall correlation between human and murine scores, important stem cell genes were similarly deregulated in both profiles.

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