Human fetal and adult hematopoietic stem cells (HSC) were obtained from fetal liver, fetal bone marrow (BM), and adult BM. These were injected into human fetal thymic implants in SCID-hu Thy/Liv mice (4-6 separate mice per HSC donor) and allowed to mature into single positive CD4+ (SP4) thymocytes over the course of 7-8 weeks. SP4 thymocytes from injected stem cells were subsequently sort-purified from thymic implants and gene expression was performed. in additon, We compared differences in fetal and adult T cells by performing whole genome profiling on sort-purified T cells (naïve CD4+ and Treg cells) from human fetal specimens (18-22 gestational weeks) and adult specimens (age 25-40 years old). Fetal and Adult Naïve CD4+ T cells phenotype: CD3+CD4+CD45RA+CCR7+CD27+, Fetal and Adult CD4+CD25+ Treg phenotype: CD3+CD4+CD25bright

HSC from fetal (age 18-22 gestational weeks) and adult (age: 19-43 year old) HLA-A2+ donors were obtained from different tissues. After injection into human fetal thymic implants (SCID-hu Thy/Liv HLA-A2-) the cells were allowed to mature into thymocytes and sorted on the basis of HLA-A2+ expression and CD3+CD4+ (SP4) expression. 3 separate thymic implants were analyzed for each group.

Four different groups were analyzed: Fetal Naïve CD4+ T cells, Adult Naïve CD4+ T cells, Fetal Treg cells, Adult Treg cells. For each group three independent donors were analyzed.

Group Set:

FL SP4, CD3+CD4+ thymocytes derived from fetal liver HSC

FBM SP4, CD3+CD4+ thymocytes derived from fetal bone marrow HSC

ABM SP4, CD3+CD4+ thymocytes derived from adult bone marrow HSC

Fetal CD4+, sort-purified CD3+CD4+CD45RA+CCR7+CD27+ naïve T cells of fetal origin

Adult CD4+, sort purified CD3+CD4+CD45RA+CCR7+CD27+ naïve T cells from adults

Fetal Treg, sort-purified CD3+CD4+CD25bright Treg cells of fetal origin

Adult Treg, sort-purified CD3+CD4+CD25bright Treg cells from adults