Extended Data Figure 1: Identification of pre-HSCs in the E11 AGM region.

a, Expression of VE-cadherin in CD31⁺CD41^lowCD45⁻ cells of the E11 AGM region. b, FACS isolation of T1 pre-HSCs on the basis of AA4.1 expression. c, Donor chimaerism in peripheral blood of recipients receiving cultures of CD31⁺CD45-CD41^lowc-Kit⁺AA4.1⁻ and CD31⁺CD45⁻CD41^lowc-Kit⁺AA4.1⁺ cells, respectively, 4 weeks after transplantation. d, Multi-lineage repopulation (8–12 weeks) in peripheral blood of secondary recipients (n = 17) receiving HSCs from three reconstituted primary recipients by single T1 pre-HSC-derived co-cultures. Donor-derived (CD45.1⁺CD45.2⁺) chimaerism in myeloid (Gr-1⁺/Mac-1⁺), B-lymphoid (B220⁺), and T-lymphoid (CD3⁺) cells are shown, and ‘a’, ‘b’ and ‘c’ indicate three different mice reconstituted by single-cell co-cultures. e, Expression of VE-cadherin in CD31⁺CD45⁺ cells of the E11 AGM region. f, FACS isolation of E11 T2 pre-HSCs with CD41. g, Donor chimaerism in peripheral blood of recipients receiving 6-day co-cultures from CD31⁺CD45⁺CD41^low population (three cells per recipient, n = 16) monitored at 4, 8, 12, and 16 weeks after transplantation. h, Multi-lineage long-term (>16 weeks) repopulation in peripheral blood, bone marrow, spleen, and thymus of primary recipients transplanted with 6-day co-cultures initiated from the CD31⁺CD45⁺CD41^low population. Donor-derived (CD45.1⁺CD45.2⁺) myeloid (Mac-1⁺/Gr-1⁺), B-lymphoid (B220⁺), and T-lymphoid (CD3⁺ or CD4⁺/CD8⁺) cells are shown in major haematopoietic organs of a representative recipient.

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