Localized SCF and IGF-1 secretion enhances erythropoiesis in the spleen of murine embryos

Abstract

Fetal spleen is a major hematopoietic site prior to initiation of bone marrow hematopoiesis. Morphologic analysis suggested erythropoietic activity in fetal spleen, but it remained unclear how erythropoiesis was regulated. To address this question, we performed flow cytometric analysis and observed that the number of spleen erythroid cells increased 18.6-fold from 16.5 to 19.5 days post-coitum (dpc). Among erythropoietic cytokines, SCF and IGF-1 were primarily expressed in hematopoietic, endothelial and mesenchymal-like fetal spleen cells. Cultures treated with SCF and/or IGF-1R inhibitors showed significantly decreased CD45-c-Kit-CD71+/-Ter119+ erythroid cells and downregulated Gata1, Klf1 and β-major globin expression. Administration of these inhibitors to pregnant mice significantly decreased the number of CD45-c-Kit-CD71+/-Ter119+ cells and downregulated β-major globin gene expression in embryos derived from these mice. We conclude that fetal spleen is a major erythropoietic site where endothelial and mesenchymal-like cells primarily accelerate erythropoietic activity through SCF and IGF-1 secretion.

Keywords: Erythropoiesis; Fetal spleen; Niche, Cytokines.

Fig. 1.. Characterization of fetal spleen and liver cells.

(A) Hematoxylin and eosin-stained sections of fetal spleen at 16.5 and 19.5 dpc. Scale bars: 100 µm. (B) Graph showing total number of Ter119+ and CD45+ cells per spleen at 16.5 and 19.5 dpc (n = 3). (C) Graph showing total number of Ter119+ and CD45+ cells per liver at 16.5 and 19.5 dpc (n = 3). (D) Immunohistochemistry staining of CD45−Ter119− non-hematopoietic fetal spleen cells. Spleen sections were prepared from ICR mouse embryos at 16.5 dpc and stained with DLK-1 (green), LYVE-1 (green), CD31 (green) and TOTO-3 (blue). Scale bars: 20 µm. (E,F) Graphs showing percentage of fetal spleen cells among non-hematopoietic cells at 16.5 and 19.5 dpc (n = 3). Data are means±standard deviation (SD). (G,H) Graphs showing percentage of fetal liver cells among non-hematopoietic cells at 16.5 and 19.5 dpc (n = 3). Data are means±standard deviation (SD). See also supplementary material Fig. S1A,B. *P<0.05.

Fig. 2.. Expression of cytokine mRNA and protein in fetal spleen and liver.

(A) Relative expression (RQ) of stem cell factor (Scf), insulin-like growth factor1 (Igf1), interleukin-3 (Il-3) and erythropoietin (Epo) mRNAs were examined in whole fetal spleen cells at 16.5 and 19.5 dpc by real-time PCR. Epo expression in 16.5 dpc whole fetal spleen served as a control. (B) Relative expression (RQ) of stem cell factor (Scf), insulin-like growth factor1 (Igf1), interleukin-3 (Il-3) and erythropoietin (Epo) mRNAs was determined in whole fetal liver at 16.5 and 19.5 dpc by real-time PCR. Epo expression in 16.5 dpc whole fetal spleen served as a control. (C,D) Amounts of SCF, IGF-1, IL-3 and EPO protein per 100 µg total protein in spleen at 16.5 and 19.5 dpc. (D) Amounts of SCF, IGF-1, IL-3 and EPO protein per 100 µg total protein in liver at 16.5 and 19.5 dpc. (E,F) Relative expression (RQ) of Scf and Igf1 mRNAs was determined by real-time PCR in hematopoietic cells (HCs), endothelial cells (ECs) and unclassified cells (UCs) sorted by flow cytometry, according to gates defined in supplementary material Fig. S1A. HCs served as controls. Scf and Igf1 mRNA expression was higher in UCs than in ECs at 16.5 dpc fetal spleen (n = 3). (G,H) Amounts of SCF and IGF-1 protein per 100,000 cells in HCs, ECs, and UCs (n = 3). See also supplementary material Fig. S1E,F. Data are means±standard deviation (SD). *P<0.05.

Fig. 3.. Characterization of unclassified cells (UCs).

(A) Analysis of the mesenchymal markers CD29, CD44, CD51, CD73, CD90.2, CD105, CD106, CD140a and CD166 among CD45−Ter119−CD31−LYVE-1− unclassified cells (n = 3). (B) Spleen sections from 16.5 dpc were stained with CD51 (green) and TOTO-3 (blue). Scale bar: 20 µm. (C) A single cell suspension was obtained from fetal spleen at 16.5 dpc. CD45−Ter119−CD31−LYVE-1−CD51+ defines CD51+ cells. Relative expression (RQ) of Scf and Igf1 mRNAs was examined by real-time PCR in CD51+ cells (n = 3). Hematopoietic cells (HCs) served as reference controls. (D) Amounts of SCF and IGF-1 protein per 100,000 cells in CD51+ cells. Expression of SCF and IGF-1 was higher in CD51+ cells than in HCs (n = 3). (E,F) Immunohistochemistry of endothelial and mesenchymal cell markers, CD31 and CD51, respectively stained with SCF and IGF-1 at 16.5 dpc. Samples were observed under confocal microscopy. CD31+/− and CD51+/− cells expressed both SCF and IGF-1. Scale bars: 20 µm. Data are mean±standard deviation (SD). *P<0.05.

Fig. 4.. Acceleration of erythropoiesis in hematopoietic cells cultured with stromal cells.

(A) Microscopic images after 24 hours of culture of hematopoietic cells with or without stromal cells. Cells that adhered to a plate after two hours were used as stromal cells. Hematopoietic cells were cultured either alone or in co-culture with stromal cells. Scale bars: left, 200 µm; right, 100 µm. (B) Graphs showing the total number of cells (left) and the percentage of viable and dead cells (right) in conditions with or without stromal cells. Both the total number and percentage of viable cells were higher among cells cultured with stromal cells (n = 3). (C) Gating strategy of flow cytometric analysis. (1) CD45−c-Kit+CD71−Ter119− defines BFU-E equivalent cells; (2) CD45−c-Kit+CD71+Ter119− defines CFU-E equivalent cells; (3) CD45−c-Kit−CD71+Ter119+ defines terminally-differentiating erythroid cells; and (4) CD45−c-Kit−CD71−Ter119+ defines mature red blood cells. See also supplementary material Fig. S3A,B. (D) Graphs showing the number of BFU-E equivalent cells, CFU-E equivalent cells, terminally-differentiating erythroid cells and mature red blood cells after 24 hours of culture with or without stromal cells. The number of terminally-differentiating erythroid cells and mature red blood cells increased in cultures including stromal cells (n = 3). Data are means±standard deviation (SD). NS, not significant. *P<0.05.

Fig. 5.. Expression of c-Kit and IGF-1R on erythroid cells.

(A) Gating strategy of flow cytometric analysis of c-Kit+ and IGF-1R+ cells during erythropoiesis. (1) CD45−CD71+Ter119− defines BFU-E and CFU-E equivalent cells, (2) CD45−CD71+Ter119+ defines terminally-differentiating erythroid cells, and (3) CD45−CD71−Ter119+ defines mature red blood cells. (B) Representative diagram of flow cytometric analysis showing cell surface expression of c-Kit and IGF-1R on erythroid cells. (C,D) Graphs showing the percentage of c-Kit+ or IGF-1R+ cells in BFU-E and CFU-E equivalent cells, terminally-differentiating erythroid cells and mature red blood cells (n = 3). c-Kit+ and IGF-1R+ cells were expressed primarily in BFU-E and CFU-E equivalent cells. Data are means±standard deviation (SD).

Fig. 6.. SCF and IGF-1 accelerate fetal spleen erythropoiesis in vitro.

(A) Microscopic images of whole fetal spleen cell cultures after 24 hours of treatment with DMSO, an SCF inhibitor, an IGF-1R inhibitor or both. Scale bars: left, 200 µm; right, 100 µm. (B) Graphs showing the total number of cells (left) and percentage of viable and dead cells (right) in culture with those inhibitors (n = 3). (C) Gating strategy of flow cytometric analysis. CD45−c-Kit−CD71+/−Ter119+ defines terminally-differentiating erythroid cells and mature red blood cells. (D) Graph showing the number of terminally-differentiating erythroid cells and mature red blood cells in culture with DMSO, the SCF inhibitor, the IGF-1R inhibitor or both (n = 3). (E) Expression of erythroid mRNAs such as Gata1, Klf1 and β-major globin in sorted CD45− cells by real-time PCR. CD45− cells from DMSO-treated cultures served as controls (n = 3). Data are means±standard deviation (SD). *P<0.05.

Fig. 7.. SCF and IGF-1 accelerate fetal spleen erythropoiesis in vivo.

(A) Gating strategy of flow cytometric analysis. CD45−c-Kit−CD71+/−Ter119+ defines terminally-differentiating erythroid cells and mature red blood cells. (B) Graph showing the number of terminally-differentiating erythroid cells and mature red blood cells in mice treated with DMSO, the SCF inhibitor, the IGF-1R inhibitor or both, two hours after injection (n = 3). (C) Relative expression (RQ) of erythroid genes such as Gata1, Klf1 and β-major globin mRNAs in CD45− cells was examined by real-time PCR. CD45− cells from mice injected with DMSO served as controls (n = 3). Data are means±standard deviation (SD). *P<0.05.