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Blood, Vol. 114, Issue 2, 268-278, July 9, 2009
Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells
Blood McKinney-Freeman et al.
114: 268
Supplemental materials for: McKinney-Freeman et al
Files in this Data Supplement:
- Figure S1. Optimization of E11.5 AGM cell derivation (JPG, 355 KB)
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(A) Representative flow cytometry analysis of E11.5 AGM-derived cells for CD34 and c-Kit expression. (B) Total cell yield/embryo of multiple AGM dissociation protocols. T, trypsin; C, collagenase; D 2.5%, 2.5% dispase; D 0.06–0.25%, 0.06–0.25% dispase. Hematopoietic colony activity of (C) unfractionated or (D) CD34+c-Kit+ AGM cells isolated with distinct dissociation protocols.
- Figure S2. CD150−CD48− AGM-derived cells can repopulate the hematopoietic compartment of secondary recipient mice (JPG, 536 KB)
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(A) CD150−CD48− AGM-derived cells were purified by FACS from E11.5 CD45.1+/CD45.2+ C57Bl/6 mice and transplanted into irradiated CD45.1 C57Bl/6 mice along with 2.5 × 105 CD45.2 C57Bl/6 WBM cells. Five–six months post-transplant, bone marrow from these primary recipients was transplanted into irradiated CD45.1 C57Bl/6 secondary recipient mice. (B) Analysis of peripheral blood of secondary recipients of CD150−CD48− AGM-derived (CD45.1+/CD45.2+) cells 18 weeks post transplant. Two independent experiments are shown. (C) Representative analysis of peripheral blood of secondary recipient of CD150−CD48− AGM-derived cells 18 weeks post-secondary transplant.
- Figure S3. Post-sort analysis of E11.5 AGM-derived cells fractionated by FACS for CD150, CD48, and CD41 (JPG, 278 KB)
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CD150−CD48−CD41+ and CD150−CD48−CD41− cells were purified from E11.5 AGM− by FACS. A representative post-sort analysis is shown.
- Figure S4. Post-sort analysis of E12.5 placenta-derived cells fractionated by FACS (JPG, 434 KB)
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(A) c-kit+CD34medCD48+ and c-kit+CD34medCD48+ cells were isolated from E12.5 placenta by FACS. A representative post-sort analysis is shown. (B) E12.5 placenta-derived cells were fractionated by FACS with respect to surface expression of CD41 and CD48 and analyzed post-sort for purity. (C) CD150+ and CD150− cells were isolated by FACS from E12.5 placenta. A representative post-sort analysis is shown.
- Figure S5. WBM and E14.5 fetal liver hematopoietic repopulating cells are CD150+CD48− (JPG, 429 KB)
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(A) Lin−CD48−CD244−CD150+ cells were purified from WBM via FACS. Pre- (upper plots) and post- (lower plots) sort analysis of WBM is shown. (B) The PB of mice transplanted with CD45.1+/CD45.2+ Lin−CD244−CD48−CD150+ WBM cells and CD45.2+ competitor WBM was analyzed for the co-expression of CD45.1, CD45.2, and mature PB lineage cell surface markers (B cells (CD19+/IgM+), T cells (CD3+ and CD4 or CD8+), and myeloid cells (Mac-1+ and/or Gr-1+)) 20 weeks post-transplant. (C) CD48−CD150+ E14.5 fetal liver-derived cells were sorted by FACS. Pre- (right plot) and post- (left plot) sort analysis of WBM is shown. (D) The PB of mice transplanted with CD45.2+ CD150+CD48− fetal liver cells and CD45.1+/CD45.2+ competitor WBM was analyzed for the co-expression of CD45.1, CD45.2, and mature PB lineage cell surface markers (B cells (CD19+/IgM+), T cells (CD3+ and CD4 or CD8+), and myeloid cells (Mac-1+ and/or Gr-1+)) 13 weeks post-transplant.
- Figure S6. EPOCH cells mediate robust hematopoietic engraftment of irradiated mice but are functionally distinct from WBM (JPG, 558 KB)
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(A) Representative images showing the expansion of iCdx4 EB-derived cells infected with retroviral HoxB4 on OP9 stroma at day-4, day-7, and day-10 post infection. Approximately 68 well-developed OP9 colonies developed per 100,000 infected EB-derived cells by day-7 post-infection. (B) Various doses of EPOCH cells were transplanted into Rag-2−∕−γc−∕− recipients subjected to 9.25 Gy of irradiation and analyzed for survival up to 60 days post-transplant. (C) Unfractionated EPOCH cells and WBM were assessed for hematopoietic colony forming activity (CFU). The total number of CFUs, as well as CFU-GEMM, CFU-G/M/GM, and CFU-E/Mk are presented. (D) 2 × 106 EPOCH cells or 2.5 × 105 WBM cells were transplanted into Rag-2−∕−γc−∕− mice conditioned with various doses of irradiation and analyzed for survival up to 6 weeks post-transplant. (E) CFUS12 of EPOCH and WBM cells after 11 gy of irradiation. (F) Representative mouse highly engrafted with EPOCH cell-derived (GFP+) B cells (CD19+/IgM+), T cells (CD3+ and CD4 or CD8+), and myeloid cells (Mac-1+ and/or Gr-1+) 16 weeks post-transplant.
- Figure S7. Representative pre- and post-sort analysis of EPOCH cells fractionated with respect to CD41, CD45, and CD34 by FACS (JPG, 868 KB)
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(A) Representative analysis of EPOCH cells fractionated by magnetic selection for cell surface CD41 or CD45 expression and examined pre-and post-fractionation by flow cytometry. (B) Representative analysis of EPOCH cells fractionated by FACS for cell surface expression of CD41 and CD45 and examined pre-and post-sort by flow cytometry. (C) Representative analysis of EPOCH cells fractionated by FACS for cell surface expression of CD41 and CD34 and examined pre-and post-sort by flow cytometry.
- Figure S8. Hematopoietic colony forming potential of EPOCH cells (JPG, 310 KB)
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(A) EPOCH cells fractionated by magnetic bead selection on the basis of cell surface expression of CD41 or CD45 were assessed for hematopoietic colony forming activity. Two independent experiments are shown. (B) EPOCH cells fractionated by FACS on the basis of CD41 and CD45 expression were assayed for hematopoietic colony forming activity. Two independent experiments are shown.
- Figure S9. Expression of α4β1 integrin and CXCR4 on ESC-HSC and embryonic–derived HSC (JPG, 401 KB)
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(A) CD41highCD34− EPOCH cells, Lin−Sca-1+c-kit+ WBM, Lin−Sca-1+c-kit+ E14.5 FL, CD41+CD34+c-kit+ E11.5 AGM, CD34+c-kit+ E12.5 placenta, and CD41+CD34+c-kit+ E9 YS were collected and analyzed side by side on the same day for the cell surface expression of α4β1 integrin and CXCR4 by flow cytometry. The data presented is representative of at least two independent experiments. (B) Because CXCR4 could not be detected on Lin-Sca-1+c-kit+CD150+CD48− WBM and Lin-Sca-1+c-kit+CD150+CD48− E14.5 by flow cytometry, these cells were purified by FACS and analyzed by RT-PCR for the expression of this molecule. The following primers for CXCR4 were used: GGC TGT AGA GCG AGT GTT GC and GTA GAG GTT GAC AGT GTA GAT. GAPDH was used to assess relative levels of gene expression. Two independent experiments are shown.
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