Fig. 4: TGFβ inhibition induces erythroid priming of HSCs.

Normalized UMI counts of selected genes, involved in TGFβ (A) and BMP (B) signaling, differentially expressed between Bthal HSCs (red) and HD HSCs (white). Results are represented as Fold respect to Ctrl (HD n = 2; Bthal n = 2). mean ± SEM is shown. C Transcript level of molecules involved in TGFβ and BMP signaling evaluated by ddPCR in MSCs. P value by two-tailed unpaired t-test; mean ± SEM is shown. D Evaluation of TGFβ1 in Bthal and HD platelet-poor plasma by enzyme-linked immunosorbent assay (ELISA) (Bthal n = 11; HD n = 9). P value by two-tailed unpaired t-test; mean ± SEM is shown. E Experimental plan for in vivo treatment of humanized mice with TGFβi inhibitor and/or BMP2. CD34⁺ mPB cells were administered to NBSGW female mice by retro-orbital injection. 9- to 10-week-old NBSGW mice were conditioned with busulfan injected intraperitoneally (15 mg/kg body weight) 24 h before transplantation. After 10 weeks, mice were treated with 10 mg/kg of TGFβ inhibitor (TGFβi) by subcutaneous injection and/or 20 ng/g of BMP2 molecule by intraperitoneal injection, twice weekly for 2 weeks. Image was created with BioRender.com. (https://BioRender.com/3tbmj37). F Frequencies of hCD45⁺ in BM from untreated animals (ctrl n = 8) and after in vivo treatment with TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. G Frequencies of CD34⁺ in hCD45+ BM from untreated animals (ctrl n = 8) and after in vivo treatment with TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. H Frequencies of HSC CD49f⁺ in hCD34⁺ from untreated animals (ctrl n = 8) and after in vivo treatment with TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. I Frequencies of Subset2 in untreated animals (ctrl n = 8) and after in vivo treatment with TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. J Frequencies of Subset 1 in untreated animals (ctrl n = 8) and after in vivo treatment with TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. K Subset1/Subset2 ratio in all groups of treated mice relative to untreated ones. Group of mice: TGFβi inhibitor (n = 8 mice), with BMP2 (n = 6 mice), or the combination of both (TGFβi + BMP2 n = 10 mice), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. L Colony assay on CD34⁺ cells purified from the untreated and treated animals. The percentage of BFUe colonies is shown. Each dot represents the mean of two dishes/mouse. Untreated animals (ctrl n = 4), TGFβi inhibitor (n = 8), BMP2 (n = 6), or the combination of both (TGFβi + BMP2 n = 5), from n = 2 independent experiments. P-values by two-tailed unpaired t-test; mean ± SEM is shown. Source data are provided as a Source Data file.