Indicated numbers of freshly isolated Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ HSCs (A–C) or their 8 day cultured progenies (D–E) from CD45.1 C57BL/6 donors were transplanted into lethally irradiated BALB/c (CD45.2) recipients without competitors (n = 4–5). HSCs were cultured in STFIA medium which allows ex vivo expansion of HSCs (Zhang et al., 2006), and there were ~200-fold increase of total cells after 8 days of culture (with 1.16 ± 0.14 × 10⁴ cultured cells derived from the input 50 cells). Panels A and D show the numbers of mice with failed or successful donor engraftment after being transplanted by indicated numbers of freshly isolated HSCs or their cultured equivalents at 16 weeks post-transplant. The 0% repopulated mice included both survived and dead ones. Panel B shows a representative flow cytometry analysis of the multi-lineage repopulation of 2500 freshly isolated HSCs at 16 weeks post-transplant. Panels C and E show multilineage contribution of indicated numbers of freshly isolated HSCs or cultured equivalent at 16 weeks post-transplant respectively (n = 4–5). Data are expressed as mean ± SEM. See also Figure S1.

(A–E) Freshly isolated 10,000 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ HSCs or their 8 day cultured progenies from CD45.1 C57BL/6 donors were transplanted into lethally irradiated BALB/c (CD45.2) recipients along with 100,000 total bone marrow cells freshly isolated from BALB/c mice as competitors. HSCs were cultured in STFIA medium which allows ex vivo expansion of HSCs. Similar results were obtained in at least two independently repeated experiments. (A–B) Representative flow cytometry plots show that 10,000 freshly isolated donor HSCs had no engraftment (left panels), whereas the cultured progeny of 10,000 input donor HSCs (right panels) had significant engraftment (54.83% CD45.1 or 49.07% H-2K^b donor chimerism in panel A or B respectively) in allogeneic recipients. (C) Summary of donor engraftment in allogeneic recipients at 4, 8, 16, and 40 weeks post-transplant (*, p < 0.05, n = 6). (D–E) Multilineage contribution of cultured cells in allogeneic recipients at 16 weeks post-transplant (n = 6). (F–G) Summary of donor engraftment at 3, 8, and 15 weeks after secondary transplantation into BALB/c mice (n = 5). Multilineage contribution of cultured cells in allogeneic recipients at 15 weeks post-transplant is shown (G). (H) MLR assay was performed in which splenocytes from C57BL/6 mice stimulated the proliferation of BALB/c T cells (bar 3) whereas splenocytes isolated from cultured C57BL/6 donor HSCs repopulated BALB/c recipients lost the ability to stimulate the proliferation of BALB/c T cells (bar 2). *, p < 0.05, n = 3. (I) Comparison of the allograft abilities of freshly isolated and cultured HSCs by limiting dilution analysis. Three types of donor cells, including freshly isolated C57BL/6 CD45.1 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻, progenies after 8-day culture in ST medium (which does not support HSC expansion), and progenies after 8-day culture in STFIA medium (which supports HSC expansion), were compared. Transplantation into lethally irradiated BALB/c mice was conducted along with 100,000 total BM competitors isolated from BALB/c mice. Limiting dilution analysis was performed and L-Calc software was used to calculate the HSC frequency (*, **, p < 0.05, compared to uncultured HSCs). Data are expressed as mean ± SEM. See also Figure S1, Figure S2, and Table S1.

(A–B) A summary of the result of flow cytometry analysis of surface expression of indicated immune molecules after 8 days of culture of HSCs in STFIA medium (*, p < 0.05, n=3–5). (C–D) CD274 surface expression was increased in cultured cells. In panel C, MFI of CD274 expression determined by flow cytometry analysis in freshly isolated and cultured HSCs are shown (*, p < 0.05, n=3–5). Shown in panel D are representative flow cytometry plots indicating 43.5% freshly isolated HSCs were CD274 positive (as CD274^low), whereas 69% of cultured cells were CD274^low and 20.0% as CD274^high. Gatings were set based on isotype controls. (E–F) Percentages of CD274^high cells and MFI of CD274 expression in different fractions of cultured HSCs. Data are expressed as mean ± SEM. See also Figure S3.

(A–C) 2 × 10⁵ freshly isolated CD45.2 CD274⁺ and CD274⁻ BM cells were transplanted, respectively, together with 2 × 10⁵ CD45.1 competitor cells into lethally irradiated congenic CD45.1 mice ((*, p < 0.05, n = 5). Panel A shows gating plots of CD274⁺ and CD274⁻ BM cells. Panel B shows peripheral blood engraftments at week 3, 10, and 16 after transplant. Panel C shows multilineage contribution of cultured cells at 16 weeks post-transplant. (D–E) 9.6 × 10⁴ sorted cultured CD45.2 CD274^high and CD274^low total cultured cells were transplanted, respectively, together with 1 × 10⁵ CD45.1 competitor cells into lethally irradiated congenic CD45.1 mice (*, p < 0.05, n = 5). Panel D shows peripheral blood engraftments at week 3, 5, and 16 after transplant. Panel E shows multilineage contribution of cultured cells at 16 weeks post-transplant. (F) All tested culture condition induces CD274 expression on the surface of HSCs (n = 3). Shown are the percentages of cells that express CD274 on their surface after 8 days of culture of HSCs in serum-free medium supplemented with SCF+TPO (ST), SCF+TPO+FGF-1 (STF), SCF+TPO+FGF-1+Angptl3 (STFA), SCF+TPO+FGF-1+IGFBP2 (STFI), and SCF+TPO+FGF-1+Angtpl3+IGFBP2 (STFIA). Data are expressed as mean ± SEM. See also Figure S4.

(A–B) CD274 does not affect repopulation of freshly isolated HSCs in syngeneic transplantation. Freshly isolated 1×10⁵ BM cells from WT or CD274 null CD45.2 C57BL/6 donors were transplanted into lethally irradiated CD45.1 C57BL/6 syngeneic recipients with 100,000 CD45.1 C57BL/6 total BM competitors (*, p < 0.05, n=5). Panel A shows donor repopulation at 3, 8, and 16 weeks post-transplant. Panel B shows multilineage contribution of cultured cells at 16 weeks post-transplant. (C–D) CD274 does not affect repopulation of cultured HSCs in syngeneic transplantation. Cultured progenies of 100 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ HSCs from WT or CD274 null CD45.2 C57BL/6 donors were transplanted into lethally irradiated CD45.1 C57BL/6 syngeneic recipients with 100,000 CD45.1 C57BL/6 total BM competitors (n = 5). Cells were cultured in STFIA medium. Panel C shows donor repopulation at 3, 12 and 17 weeks post-transplant. Panel D shows multilineage contribution of cultured cells at 17 weeks post-transplant. (E–F) CD274 enhances repopulation of cultured HSCs in competitive allogeneic transplantation. Cultured progenies of input equivalent 10,000 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ HSCs from CD45.2 C57BL/6 donors were co-transplanted with 100,000 freshly isolated BALB/c (CD45.2) BM cells into lethally irradiated BALB/c (CD45.2) recipients (*, p < 0.05, n=5). Cells were cultured in STFIA medium. This is an experiment representing two independent experiments that gave similar results. Panel E shows donor engraftment at 3, 8 and 16 weeks post-transplantation. Panel F shows multilineage contribution of cultured cells at 16 weeks post-transplant. (G) CD274 enhances repopulation of cultured HSCs in non-competitive allogeneic transplantation. Cultured progenies of input equivalent 1,000 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ WT or CD274 null HSCs were transplanted into lethally irradiated BALB/c (CD45.2) recipients without competitors. ST medium was used in culture. Shown is donor engraftment at 16 weeks post-transplant (*, p < 0.05, n = 10 – 17). (H) MLR assay was performed in which cultured WT HSCs abrogated the proliferation of allogeneic T cells (bar 2) whereas cultured CD274 null HSCs were unable to do so (bar 3). *, p < 0.05, n = 3. (I) CD274 enhances repopulation of cultured HSCs through inhibition of T cell response. Cultured progenies of input equivalent 5,000 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ WT or CD274 null HSCs were transplanted into sublethally irradiated (2.5 Gy) SCID-BALB/c (CD45.2) recipients. Cells were cultured in STFIA medium. Shown are donor engraftments at 7 and 16 weeks post-transplantation (n = 5). Data are expressed as mean ± SEM. See also Figure S5.

Freshly isolated 10,000 Lin⁻Sca-1⁺Kit⁺CD34⁻Flk2⁻ HSCs or their 8-day cultured progenies from CD45.1 FVB donors were transplanted into lethally irradiated C57BL/6/129 CD45.2 knock-in mutation at DNA-PKcs T2605 phosphorylation cluster recipients at postnatal day 12 (*, p < 0.05, n = 6–9). HSCs were cultured for 8 days in STFIA medium that allows ex vivo expansion of HSCs. When competitors were used, freshly isolated 2,000,000 – 4,000,000 Sca-1⁻ bone marrow cells isolated from FVB mice were co-transplanted. Panel A shows donor engraftment at 16 weeks post-transplant. Panel B shows multilineage contribution of cultured cells in rescued DNA-PK knock-in mice at 16 weeks post-transplant. Data are expressed as mean ± SEM.

Human cord blood Lin⁻CD34⁺CD38⁻CD90⁺ cells were cultured in serum-free medium supplemented with SCF, TPO, and Flt3-L for 8 days. (A–B) Representative plots of CD34⁺CD38⁻CD90⁺ cells that express CD274 on their surface before (A) and after culture (B). Gatings were set based on isotype controls. (C) Representative plot of CD34 and CD274 high or low staining in cultured human cells. (D) Summary of percentages of CD274⁺ and CD274^high cells in uncultured and cultured human cells (*, p < 0.05, n = 5). (E) MFI of CD274 expression determined by flow cytometry analysis in freshly isolated and cultured human cells (*, p < 0.05, n = 5). (F) MLR assay was performed in which cultured human cord blood HSCs abrogated the proliferation of allogeneic T cells (bar 2) whereas anti-CD274 antibody reversed the inhibitory effect of cultured human cord blood HSCs (bar 3). *, p < 0.05, n = 6. Data are expressed as mean ± SEM. See also Figure S6.