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p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, Villeval JL, Louache F, Gonin P, Debili N, Plo I, Vainchenker W, Gilles L, Raslova H - Stem Cell Reports (2014)

Bottom Line: We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition.Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion.This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U1009, Equipe labellisée Ligue Nationale contre le Cancer, 114 rue Edouard Vaillant, 94805 Villejuif, France; University Paris Sud, 114, rue Edouard Vaillant, 94805 Villejuif, France; Gustave Roussy, IFR54, 114, rue Edouard Vaillant, 94805 Villejuif, France; University Paris Diderot, 5 rue Thomas-Mann, 75205 Paris, France.

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Absence of p19INK4d Leads to a Progressive Age-Dependent Increase in the Number of MKs in BM and Spleen of Mice(A and B) Number of MK-Ps derived from total BM cells (A) and total spleen cells (B) of 8- to 10-week-old (n = 3) and 20- to 25-week-old (n = 4) mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(C–F) Counts of MKs identified by vWF staining in BM (C) and spleen (D) of 8- to 10-week-old (n = 3), 20- to 25-week-old (n = 4) and in >91-week-old (n = 6) mice. The number of MKs was determined on equal areas of three independent image frames for each mouse. Representative pictures are shown for BM (E) and spleen (F).(G) Cell cycle was measured by PI staining (middle panel) within the MK (CD41+) population (left panel). Total and ≥8N MK ploidy level (right panel) was calculated for 20- to 25-week-old and >91-week-old mice.(H) Mean ploidy level calculated for ≥8N MK.(I) Percentage of 2N/4N MK cells in BM of 20- to 25-week-old and >91-week-old mice. Data represent mean ± SEM (n = 4) for 20- to 25-week-old and (n = 3) for >91-week-old mice.(J and K) Number of MK-P derived from total BM cells (J) and total spleen cells (K) in 8-month-old transplanted WT and KO mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(L and M) Counts of MKs identified by vWF staining in BM (L) and spleen (M) in 8-month-old transplanted WT and KO mice (n = 5). The MK number was determined on equal areas of three independent image frames for each mouse.KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S3.
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fig5: Absence of p19INK4d Leads to a Progressive Age-Dependent Increase in the Number of MKs in BM and Spleen of Mice(A and B) Number of MK-Ps derived from total BM cells (A) and total spleen cells (B) of 8- to 10-week-old (n = 3) and 20- to 25-week-old (n = 4) mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(C–F) Counts of MKs identified by vWF staining in BM (C) and spleen (D) of 8- to 10-week-old (n = 3), 20- to 25-week-old (n = 4) and in >91-week-old (n = 6) mice. The number of MKs was determined on equal areas of three independent image frames for each mouse. Representative pictures are shown for BM (E) and spleen (F).(G) Cell cycle was measured by PI staining (middle panel) within the MK (CD41+) population (left panel). Total and ≥8N MK ploidy level (right panel) was calculated for 20- to 25-week-old and >91-week-old mice.(H) Mean ploidy level calculated for ≥8N MK.(I) Percentage of 2N/4N MK cells in BM of 20- to 25-week-old and >91-week-old mice. Data represent mean ± SEM (n = 4) for 20- to 25-week-old and (n = 3) for >91-week-old mice.(J and K) Number of MK-P derived from total BM cells (J) and total spleen cells (K) in 8-month-old transplanted WT and KO mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(L and M) Counts of MKs identified by vWF staining in BM (L) and spleen (M) in 8-month-old transplanted WT and KO mice (n = 5). The MK number was determined on equal areas of three independent image frames for each mouse.KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S3.

Mentions: MKs play an important role in the BM niche via secretion of cytokines such as TGF-β1 and by cell-cell interactions. This led us to perform a detailed study of the MK lineage in p19INK4d−/− mice during aging, as this is a biological process that is known to modify HSC properties. While no differences were observed in MK-P numbers of 8- to 10-week-old mice compared with WT mice, the frequency of MK-Ps at 20–25 weeks was increased 2.3-fold in the BM and 6.1-fold in the spleen of p19INK4d−/− mice (Figures 5A and 5B). In contrast, no differences were observed for all other BM myeloid progenitors during aging (Figures S3A and S3B). In peripheral blood, a significant increase in red blood cells and lymphocytes detected in WT mice was not observed in p19INK4d−/− mice (Figures S3C–S3F). In contrast, the slight increase in platelet counts observed in young p19INK4d−/− animals was more pronounced (1.35-fold) in p19INK4d−/− mice that were older than 91 weeks (Figure S3F).


p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Hilpert M, Legrand C, Bluteau D, Balayn N, Betems A, Bluteau O, Villeval JL, Louache F, Gonin P, Debili N, Plo I, Vainchenker W, Gilles L, Raslova H - Stem Cell Reports (2014)

Absence of p19INK4d Leads to a Progressive Age-Dependent Increase in the Number of MKs in BM and Spleen of Mice(A and B) Number of MK-Ps derived from total BM cells (A) and total spleen cells (B) of 8- to 10-week-old (n = 3) and 20- to 25-week-old (n = 4) mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(C–F) Counts of MKs identified by vWF staining in BM (C) and spleen (D) of 8- to 10-week-old (n = 3), 20- to 25-week-old (n = 4) and in >91-week-old (n = 6) mice. The number of MKs was determined on equal areas of three independent image frames for each mouse. Representative pictures are shown for BM (E) and spleen (F).(G) Cell cycle was measured by PI staining (middle panel) within the MK (CD41+) population (left panel). Total and ≥8N MK ploidy level (right panel) was calculated for 20- to 25-week-old and >91-week-old mice.(H) Mean ploidy level calculated for ≥8N MK.(I) Percentage of 2N/4N MK cells in BM of 20- to 25-week-old and >91-week-old mice. Data represent mean ± SEM (n = 4) for 20- to 25-week-old and (n = 3) for >91-week-old mice.(J and K) Number of MK-P derived from total BM cells (J) and total spleen cells (K) in 8-month-old transplanted WT and KO mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(L and M) Counts of MKs identified by vWF staining in BM (L) and spleen (M) in 8-month-old transplanted WT and KO mice (n = 5). The MK number was determined on equal areas of three independent image frames for each mouse.KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S3.
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fig5: Absence of p19INK4d Leads to a Progressive Age-Dependent Increase in the Number of MKs in BM and Spleen of Mice(A and B) Number of MK-Ps derived from total BM cells (A) and total spleen cells (B) of 8- to 10-week-old (n = 3) and 20- to 25-week-old (n = 4) mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(C–F) Counts of MKs identified by vWF staining in BM (C) and spleen (D) of 8- to 10-week-old (n = 3), 20- to 25-week-old (n = 4) and in >91-week-old (n = 6) mice. The number of MKs was determined on equal areas of three independent image frames for each mouse. Representative pictures are shown for BM (E) and spleen (F).(G) Cell cycle was measured by PI staining (middle panel) within the MK (CD41+) population (left panel). Total and ≥8N MK ploidy level (right panel) was calculated for 20- to 25-week-old and >91-week-old mice.(H) Mean ploidy level calculated for ≥8N MK.(I) Percentage of 2N/4N MK cells in BM of 20- to 25-week-old and >91-week-old mice. Data represent mean ± SEM (n = 4) for 20- to 25-week-old and (n = 3) for >91-week-old mice.(J and K) Number of MK-P derived from total BM cells (J) and total spleen cells (K) in 8-month-old transplanted WT and KO mice cultured in fibrin clots. Experiments were performed in triplicate for each biological replicate.(L and M) Counts of MKs identified by vWF staining in BM (L) and spleen (M) in 8-month-old transplanted WT and KO mice (n = 5). The MK number was determined on equal areas of three independent image frames for each mouse.KO, p19INK4d−/−. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, unpaired t test. See also Figure S3.
Mentions: MKs play an important role in the BM niche via secretion of cytokines such as TGF-β1 and by cell-cell interactions. This led us to perform a detailed study of the MK lineage in p19INK4d−/− mice during aging, as this is a biological process that is known to modify HSC properties. While no differences were observed in MK-P numbers of 8- to 10-week-old mice compared with WT mice, the frequency of MK-Ps at 20–25 weeks was increased 2.3-fold in the BM and 6.1-fold in the spleen of p19INK4d−/− mice (Figures 5A and 5B). In contrast, no differences were observed for all other BM myeloid progenitors during aging (Figures S3A and S3B). In peripheral blood, a significant increase in red blood cells and lymphocytes detected in WT mice was not observed in p19INK4d−/− mice (Figures S3C–S3F). In contrast, the slight increase in platelet counts observed in young p19INK4d−/− animals was more pronounced (1.35-fold) in p19INK4d−/− mice that were older than 91 weeks (Figure S3F).

Bottom Line: We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition.Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor β1 secretion.This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U1009, Equipe labellisée Ligue Nationale contre le Cancer, 114 rue Edouard Vaillant, 94805 Villejuif, France; University Paris Sud, 114, rue Edouard Vaillant, 94805 Villejuif, France; Gustave Roussy, IFR54, 114, rue Edouard Vaillant, 94805 Villejuif, France; University Paris Diderot, 5 rue Thomas-Mann, 75205 Paris, France.

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Related in: MedlinePlus