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SC_12-0246_sm_supplFigure1B.tif1067KFig S1: DL-4 cells upregulate CD45RA and CXCR4 during culture A: Phenotypic analysis of CD34 and CD7 expression by DL-4 cells harvested after 3, 7 and 14 days of culture (upper line). The surface expression of CD45RA (middle line) and CXCR4 (lower line) was tested within the CD34+/CD7−, CD34+/CD7+ (ETP) and CD34−/CD7++ (proT) subsets. RCN: relative cell number. B: Flow cytometric analysis of surface CXCR4 expression, as measured by gating on CD34− /CD7++, CD34−/CD7++/CD5+ and CD34−/CD7++/CD56+ subsets.
SC_12-0246_sm_supplFigure1A.tif1518KFig S1: DL-4 cells upregulate CD45RA and CXCR4 during culture A: Phenotypic analysis of CD34 and CD7 expression by DL-4 cells harvested after 3, 7 and 14 days of culture (upper line). The surface expression of CD45RA (middle line) and CXCR4 (lower line) was tested within the CD34+/CD7−, CD34+/CD7+ (ETP) and CD34−/CD7++ (proT) subsets. RCN: relative cell number. B: Flow cytometric analysis of surface CXCR4 expression, as measured by gating on CD34− /CD7++, CD34−/CD7++/CD5+ and CD34−/CD7++/CD56+ subsets.
SC_12-0246_sm_supplFigure2C.tif358KFig S2: DL-4 cells can reconstitute the thymus when transferred into irradiated adult NOD/SCID/γc−/− (NSG) mice and into non-irradiated, newborn NSG mice and accelerates thymopoesis in vivo A: CD34+/CD7− and ETP /proT1 cells sorted from a 7-day DL-4 culture were transplanted into 4 week old irradiated NSG. Thymic reconstitution was assessed after 8 weeks by flow cytometry. CD4, CD8, CD3 and TCRαβ expression was studied within hCD45+/7AAD- cells. B: Irradiated adult NSG mice (upper group) and non-irradiated newborn NSG mice (lower group) were injected with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells. Thymus reconstitution was assessed 8 weeks (in adult recipients) or 4 weeks (in newborn recipients) after transplantation. The figure shows the flow cytometry analysis for thymic reconstitution as described above. C: A total of 29 non-irradiated newborn NSG were transplanted with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells in two independent transplantation series. Results of the thymic reconstitution kinetics in DL-4 versus untreated CD34+ cells are described in Table 4. Phenotypic images of representative thymi recovered from DL-4 recipients at 7 and 14 days post-transplant are shown.
SC_12-0246_sm_supplFigure2B.tif645KFig S2: DL-4 cells can reconstitute the thymus when transferred into irradiated adult NOD/SCID/γc−/− (NSG) mice and into non-irradiated, newborn NSG mice and accelerates thymopoesis in vivo A: CD34+/CD7− and ETP /proT1 cells sorted from a 7-day DL-4 culture were transplanted into 4 week old irradiated NSG. Thymic reconstitution was assessed after 8 weeks by flow cytometry. CD4, CD8, CD3 and TCRαβ expression was studied within hCD45+/7AAD- cells. B: Irradiated adult NSG mice (upper group) and non-irradiated newborn NSG mice (lower group) were injected with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells. Thymus reconstitution was assessed 8 weeks (in adult recipients) or 4 weeks (in newborn recipients) after transplantation. The figure shows the flow cytometry analysis for thymic reconstitution as described above. C: A total of 29 non-irradiated newborn NSG were transplanted with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells in two independent transplantation series. Results of the thymic reconstitution kinetics in DL-4 versus untreated CD34+ cells are described in Table 4. Phenotypic images of representative thymi recovered from DL-4 recipients at 7 and 14 days post-transplant are shown.
SC_12-0246_sm_supplFigure2A.tif456KFig S2: DL-4 cells can reconstitute the thymus when transferred into irradiated adult NOD/SCID/γc−/− (NSG) mice and into non-irradiated, newborn NSG mice and accelerates thymopoesis in vivo A: CD34+/CD7− and ETP /proT1 cells sorted from a 7-day DL-4 culture were transplanted into 4 week old irradiated NSG. Thymic reconstitution was assessed after 8 weeks by flow cytometry. CD4, CD8, CD3 and TCRαβ expression was studied within hCD45+/7AAD- cells. B: Irradiated adult NSG mice (upper group) and non-irradiated newborn NSG mice (lower group) were injected with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells. Thymus reconstitution was assessed 8 weeks (in adult recipients) or 4 weeks (in newborn recipients) after transplantation. The figure shows the flow cytometry analysis for thymic reconstitution as described above. C: A total of 29 non-irradiated newborn NSG were transplanted with 5 × 105 sorted DL-4 progenitors or 1.5 × 105 non-cultured CD34+ cells in two independent transplantation series. Results of the thymic reconstitution kinetics in DL-4 versus untreated CD34+ cells are described in Table 4. Phenotypic images of representative thymi recovered from DL-4 recipients at 7 and 14 days post-transplant are shown.
SC_12-0246_sm_supplFigure3.pdf736KFigure S3: DL-4 cells give rise to polyclonal thymocytes in recipient mice TCRβ VJ rearrangement patterns were analyzed for a number of reconstituted thymuses from adult and newborn NSG recipients. A) The TCRβ VJ recombination pattern in a representative thymus reconstituted with DL-4 cells. The image represents the result of an ImmunTraCkeR® Multiplex PCR that detects all recombination events between the TCRβ V and TCRβ J loci. B) The diagram shows the combinatorial diversity of the TCRβ VJ rearrangements in reconstituted thymuses from mice injected with DL-4-cells (left column), non-cultured CD34+ cells (middle column) and control T lymphocytes from healthy donors (right column). Each dot indicates the combinatorial diversity of an individual sample. Bars indicate median values.
SC_12-0246_sm_supplFigure4.tif1486KFigure S4: DL-4 cells give rise to peripheral T-cells in recipient mice and specifically reconstitute the T-cell compartment A) Mature T-cells were found in the spleen of 4 out of 12 DL-4 cell recipients, 1 out of 7 noncultured CD34+ cell recipient and 4 out of 6 mice co-transplanted with DL-4 and CD34+ cells. The Figure shows a flow cytometry analysis of CD3, CD4 and TCRαβ expression in a representative spleen from each type of transplantation. B) Mice were co-injected with HLA-A2- DL-4 cells and HLA-A2+ untreated CD34+ CB cells. The upper panel shows an experiment gated on hCD45+ spleen cells. HLA-A2 expression was examined by gating on the entire hCD45+ spleen cell population (left histogram) and on the splenic CD3+/TCRαβ+ T-cells in particular (right histogram). The flow cytometry analysis pictured is representative of three different mice.
SC_12-0246_sm_supplTable1.pdf55KSupplementary Table 1
SC_12-0246_sm_supplTable2.pdf19KSupplementary Table 2

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