Figure 6.

Refinement of the E14.5 iST:LT-RU immunophenotype

(A) Heatmap of marker genes for each E14.5 FL LSK transcriptional cluster.

(B) Intra-cluster mean average (MA) plots of iLT-HSCs vs. iST-HSCs in cluster 4 (i) and cluster 7 (ii).

(C) MA plots of iLT-HSCs in cluster 4 vs. cluster 7 (i) and iST-HSCs in cluster 4 vs. cluster 7 (ii).

(D) (i) Heatmap of differentially expressed genes between iST-HSCs in cluster 4 vs. cluster 7. (ii) Representative flow cytometry plots of PTPRC (CD45) expression by iLT-HSCs and iST-HSCs. n = 14 embryos from 3 litters, 3 experimental days. Values, mean ± SEM.

(E and F) Reconstitution from 100 iLT-HSC CD45⁺, 100 iST-HSC CD45⁺, or 100 iST-HSC CD45^low/- cells transplanted into lethally irradiated recipients. Plts, Ery, and Leuco lineages were analyzed after 2 (E) or 16 (F) weeks. ns, not statistically significant. ^∗, p < 0.05. ^∗∗∗∗, p < 0.00005. iLT-HSC CD45⁺, n = 7 recipients, donor cells pooled from 2 litters, 2 experimental days. iST-HSC CD45⁺, n = 9 recipients, donor cells pooled from 3 litters, 3 experimental days. iST-HSC CD45^low/-, n = 8 recipients, donor cells pooled from 3 litters, 3 experimental days.

(G) Secondary transplantations using 1/10^th doses of primary recipient bone marrow that had received a 100 cell dose of iST-HSC CD45⁺. Peripheral blood Plt, Ery, and Leuco lineages were analyzed after 16 weeks n = 3 secondary recipients from 2 primary recipients. Values, frequency of secondary reconstitution. For (E)–(G), donor-derived reconstitution (Reconstitution %) was determined using donor-specific GFP expression.