Extended Data Figure 5: FACS analysis after secondary transplantation of HE-7TF cells.

Human CD34⁺ cells were obtained by magnetic cell isolation from bone marrow of primary recipients of HE-7TF cells, then 3,000 cells (a, b) or 1,000 cells (c) were intrafemorally transplanted into secondary recipients. Multi-lineage engraftment in bone marrow from a representative recipient mouse is shown; a, 8 weeks; b, 14 weeks; c, 16 weeks. Specific mice analysed are indicated in Fig. 2b. d, Limiting dilution assay of HE-7TF cells after secondary transplantation. CD34⁺ cells were isolated from bone marrow of primary recipients, and either 1,000 or 3,000 cells were transplanted into secondary recipients. Multi-lineage engrafted recipients were counted as response. Confidence interval of 1/(stem-cell frequency) was calculated by ELDA (http://bioinf.wehi.edu.au/software/elda/) according to Poisson distribution. A limiting dilution assay of cord blood was used as reference. e, Transgene detection in engrafted cells of secondary recipients of HE-7TF cells. Recipient numbers are from Fig. 2b (numbers 31 and 35). f, Bone marrow chimaerism of primary mouse engrafted with 5-TF^Poly at 12 weeks. Human CD45⁺ bone marrow of engrafted NSG was analysed for HSPCs (CD34⁺CD38⁻), nucleated erythroid (GLY-A⁺SYTO60⁺), enucleated erythroid (GLY-A⁺SYTO60⁻), neutrophils (PECAM⁺CD15⁺), B cells (IgM⁺CD19⁺), B progenitor cells (IgM⁻CD19⁺), B lymphocytes (IgM⁻CD19⁺CD38⁺⁺), and T cells (CD3⁺/CD4, CD8). g, Representative FACS plots of bone marrow engrafted with human cord blood HSCs are shown at 10 and 12 weeks.