Extended Data Figure 8: Characterizations of differentiated cells: analysis of definitive erythropoiesis by relative quantification of globin transcripts.

a, Human GLY-A⁺ cells were isolated from lysed bone marrow (to exclude enucleated cells) and analysed by qRT–PCR to quantify (b) HBE, (c) HBG, and (d) HBB genes. CB, GLY-A⁺ erythroid cells from cord-blood-engrafted in NSG bone marrow; 7 TF HSPCs, GLY-A⁺ erythroid cells from seven transcription factor HSPC-engrafted in NSG bone marrow; 5F, GLY-A⁺ erythroid cells from hPSCs transduced with ERG, RORA, HOXA9, SOX4, and MYB⁷. Analysis of T-cell receptor diversity in engrafted T cells. e, Flow cytometric phenotyping of T cells from engrafted HE-7TF cells. Thymus was collected at 8 weeks and analysed for T-cell markers (CD4, CD8, CD3, TCRαβ, and TCRγδ). TCR phenotyping of the CD3⁺ population is shown on the right. One out of three recipients showed the presence of TCRγδ. Three thymic engrafted mice from independent experiments each. f–h, TCR rearrangement of thymocytes from cord blood CD34⁺ and HE-7TF engrafted in NSG. CD3⁺ T cells were isolated from NSG mice engrafted with (f) cord blood HSCs or (g) HE-7TF cells. Purified DNA was subjected to next-generation sequencing of the CDR3 using immunoSEQ (Adaptive Biotechnology) and analysed with the immunoSEQ Analyzer software (Adaptive Biotechnology). A high degree of combinatorial diversity in the V-gene segment usage was observed in CDR3 length, following a standard Gaussian distribution. h, Frequency of clonotype of T cells. i, Flow cytometric phenotyping of spleens from engrafted HE-7TF cells versus cord blood (j). Spleens were collected at 8 weeks and human CD45⁺ cells were analysed for T-cell markers (TCRβ, CD4, CD45RO, and CD45RA), and B-cell marker (CD19).

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