Extramedullary hematopoiesis contributes to enhanced erythropoiesis during pregnancy via TGF-β signaling

Red blood cells are the predominant cellular component in human body, and their numbers increase significantly during pregnancy due to heightened erythropoiesis. CD71+ erythroid cells (CECs) are immature red blood cells, encompassing erythroblasts and reticulocytes, constitute a rare cell population primarily found in the bone marrow, although they are physiologically enriched in the neonatal mouse spleen and human cord blood. Presently, the mechanisms underlying the CECs expansion during pregnancy remain largely unexplored. Additionally, the mechanisms and roles associated with extramedullary hematopoiesis (EMH) of erythroid cells during pregnancy have yet to be fully elucidated. In this study, our objective was to examine the underlying mechanisms of erythroid-biased hematopoiesis during pregnancy. Our findings revealed heightened erythropoiesis and elevated CECs in both human and mouse pregnancies. The increased presence of transforming growth factor (TGF)-β during pregnancy facilitated the differentiation of CD34+ hematopoietic stem and progenitor cells (HSPCs) into CECs, without impacting HSPCs proliferation, ultimately leading to enhanced erythropoiesis. The observed increase in CECs during pregnancy was primarily attributed to EMH occurring in the spleen. During mouse pregnancy, splenic stromal cells were found to have a significant impact on splenic erythropoiesis through the activation of TGF-β signaling. Conversely, splenic macrophages were observed to contribute to extramedullary erythropoiesis in a TGF-β-independent manner. Our results suggest that splenic stromal cells play a crucial role in promoting extramedullary erythropoiesis and the production of CECs during pregnancy, primarily through TGF-β-dependent mechanisms.


Isolation of mouse PBMCs
Peripheral blood samples were collected from the vena orbitalis, heparinized, and purified by centrifugation over the Ficoll-Paque Premium (1.077 g/mL density gradient medium; Cytiva, Piscataway, NJ, USA) at 400 g for 30 min at 20℃.PBMCs were collected and washed.The cells were filtered through a 40 µm cell strainer (Thermo Fisher Scientific, Waltham, MA, USA) and re-suspended for further analysis.

Isolation of mouse spleen mononuclear cells and macrophages
Mouse spleen was collected, cut into small pieces and mashed through a 40 μm cell strainer to obtain single-cell suspensions.Mononuclear cells were then enriched by lysing red cells using a red cell lysis buffer (Biolegend, San Diego, CA, USA).In some experiments, spleen macrophages were isolated from the spleen mononuclear cells.In brief, cells were stained with fluorescence-conjugated Abs against CD45, CD3 and F4/80 (BioLegend), and CD45 + CD3 - F4/80 + cells were sorted using the Sony LE-SH800ZBP flow cytometer (Tokyo, Japan).Sorted cells were analyzed using flow cytometry, and the cell purity was > 99%.

Isolation of mouse bone marrow mononuclear cells
Mouse bone marrow cells were extracted from the marrow cavities of mouse femurs and humeri by repeated flushing PBS into the marrow cavities.Mononuclear cells were then collected and enriched by lysing red cells using a red cell lysis buffer (BioLegend).The cells were filtered through a 40 µm cell strainer and re-suspended for further analysis.

Isolation of mouse decidua mononuclear cells
Mouse uterine tissues were collected, washed, cut into small pieces, and digested using 1 mg/mL collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) and 0.1 mg/mL DNase I (Roche, Basel, Switzerland) at 37℃ for about 20 min in a shaking water bath.When single or clumps of cells were observed under the microscope, released cells were separated from undigested tissue pieces by filtering through a 40 µm cell strainer.Mononuclear cells were purified over the Ficoll-Paque Premium by centrifugation at 400 g for 30 min at 20℃.Mononuclear cells were collected, washed, and re-suspended for further analysis.

Quantitative RT-PCR
Total tissue RNA was extracted using the NucleoSpin RNA Plus kit (Macherey-Nagel, Düren, Germany) according to the manufacturer's protocol.The yield and purity of total RNA were determined using the Nanodrop2000 kit (Thermo Fisher Scientific).Subsequently, 1 µg of total RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.RT-PCR was performed using the iQ SYBR Green Supermix Kit (Bio-Rad) with the StepOnePlus Real-time PCR System (Applied Biosystems, Waltham, MA).The primers were listed in Additional file 1: Table S2.The mRNA levels were measured with GAPDH (Sangon, Shanghai, China) as the internal reference.

Figure S1 .
Figure S1.Comparison of the numbers of circulating HPSCs and their subgroups, and their components in the peripheral blood of non-pregnant and pregnant women.(A)A graphical summary of the numbers of HSPCs and their subgroups in the peripheral blood of non-pregnant and pregnant women were presented.Graphical summaries of the percentages of HSPC components in the peripheral blood of non-pregnant women (B) and pregnant women (C) were shown.Data were analyzed using independent Student's t-test.Results were expressed as mean ± SD. # P < 0.05 and * P < 0.01 vs the control group.Non-P: non-pregnant women; P: pregnant women.

Figure S2 .
Figure S2.Altered composition of HSPC subsets in pregnant mice.Representative flow cytometric scatter plots of HSPCs and their subgroups in the bone marrow of non-pregnant (A) and pregnant mice (B) were presented.Graphical summaries of the percentages of HSPCs and their subgroups in mouse bone marrow (C) and spleen (D) were shown.(E) The spleens of non-pregnant and pregnant mice were shown.Data were analyzed using independent Student's t-test.Results were expressed as mean ± SD. # P < 0.05 vs. the control group.Non-P: nonpregnant mice; P: pregnant mice.

Figure S3 .
Figure S3.Effects of pregnancy-related hormones on the generation of CECs from CD34 + HSPCs.Representative flow cytometry scatter plots of HSPCs-derived CECs treated with vehicle (present as Control group) (A) or different concentrations of estradiol (B), progesterone (C), and β-hCG (D) were shown.Graphical summaries of the percentages of CECs from UBMC-derived HSPCs with vehicle or different concentrations of pregnancy-related hormones (E) were presented.Data were analyzed using one-way ANOVA.Results were expressed as mean ± SD.E2: estradiol.Prog: progesterone.

Figure S4 .
Figure S4.TGF- downstream pathways during TGF--induced erythropoiesis.Relative mRNA levels of Smad2 (A), Smad3 (B), and Smad4 (C) expressed by TGF-β-treated CD34 + HSPCs cultured under erythroid differentiation conditions.Cells treated with vehicle present as Control group.Data were analyzed using one-way ANOVA.Results were expressed as mean ± SD. # P < 0.05 and * P < 0.01 vs. the control group.

Figure S5 .
Figure S5.Effects of TGF-β on the proliferation of UBMC-derived CD34 + HSPCs.Representative flow cytometric histograms of Alex Flour 488 + cells from UBMC-derived CD34 + HSPCs in proliferation media supplemented with different concentrations of TGF-β at 24 h (A) or 48 h (C) were shown.Cells treated with vehicle present as Control group.Graphical summaries of the of Alex Flour 488 + cells from UBMC-derived HSPCs in proliferation media supplemented with different concentrations of TGF-β at 24 h (B) or 48 h (D) were presented.Data were analyzed using one-way ANOVA.Results were expressed as mean ± SD.