A subset of patients with MDS express high levels of RSPO3. (A) Representative FACS plots and corresponding bar graph quantifying the proportion of CD71^–Ter119⁺ RBCs after in vitro culturing in the absence or presence of indicated growth factors; n = 3 to 9 per condition. (B) Bar graph quantifying the proportion of viable CD45RA⁺IgM^– B-progenitors from control animals after culturing for 48 hours in the absence or presence of indicated growth factors; n = 4 per condition. (C) Expression of RSPO2 and RSPO3 in bone marrow (BM) of n = 13 control donors and n = 29 patients with MDS. Gene expression was obtained in the form of normalized reads per kilobase gene model per million total reads (nRPKM). (D) Circulating RSPO3 levels in plasma from control donors and patients with MDS; n = 9 per group. (E) Representative images of ISH for RSPO3 transcript distribution in BM core biopsy samples from n = 2 control donors and n = 12 patients with MDS. Rectangles mark regions shown at higher magnification; arrow points at a hematopoietic cell with an RSPO3 transcript. (F) Bar graph quantifying the expression of RSPO3 in normal BM and MDS BM. ISH-positive signal is normalized to hematoxylin areas. Data are represented as mean ± standard error of the mean. One-way analysis of variance followed by Dunnett’s test for multiple comparison (panels A and B), Mann-Whitney test for (panels C and D). Scale bar, 10 μm. FITC, fluorescein isothiocyanate; ND, normal donor; n.s., not significant (P > .05); PE, phycoerythrin.