Fig 2: CLEC14A-specific CAR design, expression, and function.(A) Schematic representation of a recombinant retroviral vector encoding CLEC14A-specific CARs. Retroviral CAR vector (pMP71) coexpresses a truncated CD34 marker gene and an scFv fragment/CD3? chain chimeric receptor with a CD28 costimulatory domain. Expression is driven from the LTR promoter, and the 2A peptide linker ensures equimolar expression of both molecules. (B) Recombinant retroviral expression vectors encoding CARs CAR3.28z and CAR5.28z (with scFv fragments from CLEC14A-specific monoclonal antibodies CRT3 or CRT5, respectively) were used to transduce human T cells. CAR expression was detected by staining for the CD34 marker. Percentage values show proportion of cells stained for CD34 compared with mock-transduced T cells. (C) Transduced T cells stained for expression of CAR using CLEC14A-Fc (percentage values show specific binding of CLEC14A-Fc having subtracted background staining with Fc alone). (D–F) Human T cells engineered to express putative CLEC14A-specific CARs (or mock-transduced T cell controls) were tested using an ELISA for IFN-? production for response to plate-bound recombinant CLEC14A-Fc (extracellular domain) fusion protein (or Fc alone) (D), CHO cells engineered to express full-length human CLEC14A (or CHO transduced with vector alone) (responder/stimulator [R/S] ratio = 6:1) (E), and HUVECs naturally expressing CLEC14A (or medium alone). (R/S ratio = 1:1) (F). In all cases, the different CAR T cell lines were diluted with mock T cells to equalize for transduction efficiency. Cells were stimulated for 18 hours before testing for IFN-? production. Results of ELISAs show data from 6–7 repeat experiments, having subtracted background responses of T cells alone. All P values shown were calculated using a Wilcoxon matched-pairs signed rank test.

Fig 3: Depletion of YAP/TAZ protein by DH-impaired erythroid cell proliferation and maturation. A Expression of YAP and TAZ after 10 µM lysophosphatidic acid (LPA; a YAP/TAZ activator) and dobutamine hydrochloride (DH; a YAP/TAZ inhibitor) treatment of PB-CD34+ HSC-derived erythroblasts for 11 days. Relative levels to ß-actin were labeled in red. B Fold increase of cells during erythroid differentiation from PB-CD34+ HSCs after treatment with LPA (green) and DH (red) when compared to control (black) (n = 5). C Cell apoptosis of PB-CD34+ HSC-derived erythroid cells at days 15, and 18 after LPA and DH treatment, as analyzed by Annexin V and 7-AAD staining (n = 3). D Representative cell morphology during erythroid differentiation from PB-CD34+ HSCs showed morphological delay around day 15, and most of the remaining cells were erythroblasts (black arrow) at day 18 after DH treatment. E Percentage of mature erythrocytes and erythroblasts at the terminal stage of differentiation (day 18). At least 500 cells were counted in each group (n = 9). F Expression of the erythroid-specific genes KLF1 and GATA1, after LPA and DH treatment for 8 days (n = 3). Data represent the mean ± SEM, *p < 0.5, **p < 0.01, ***p < 0.001. Scale bar, 20 µm

Fig 4: Human bone marrow hematopoietic cell expansion post MEISi treatments. (A) the percentage of CD34+ hBM MNC content, (B) the percentage of CD133+ hBM MNC content, (C) the percentage of CD90+hBM MNC content D) the percentage of CD34+CD38- hBM MNC content, (E) the percentage of CD34+CD133+CD90+ hBM MNC content, (F) the percentage of CD34+CD133+CD90+CD38- hBM MNC post 7 days of treatment with MEISi-1 and MEISi-2 compared to the cells treated with DMSO. n = 3, *p < 0.05.

Fig 5: Expression of YAP and TAZ in CD34+ HSC-derived erythroblasts. A Schematic of erythroid differentiation from human CD34+ HSCs in the three-stage erythroid culture system. B Wright’s staining of erythroid cells during differentiation of mobilized-peripheral blood (PB)- and cord blood (CB)-CD34+ HSCs showing the morphologic change from CD34+ HSC to pro-erythroblasts on day 8, erythroblasts on days 11–15, and mature erythrocytes on days 18 and 20 in in vitro culture, respectively. C Percentage of mature erythrocytes derived from PB- and CB-CD34+ HSCs in the three-stage erythroid culture system at the end of culture. At least 500 cells were counted in each group (n = 4). D, F Western blot analysis of total YAP and TAZ expression at days (D) 8, 11, 15, and 18 of erythroid-differentiated cells derived from PB- and CB-CD34+ HSCs, respectively. Relative expression levels of YAP and TAZ to ß-actin as measured by Image J software were labeled in red (PB and CB, n = 5, 3, respectively). E, G Fold intensity of YAP and TAZ were analyzed compared to CD34+ cells. Data represent the mean ± standard error of the mean (SEM), *p < 0.5. Scale bar, 20 µm