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. 2024 Aug 1;19(8):1189–1204. doi: 10.1016/j.stemcr.2024.07.003

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© 2024 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Discovery of large numbers of LT-RUs in the iST-HSC population

(A) 1,000 E14.5 iLT-HSCs (purple) or iST-HSCs (blue) were transplanted into lethally irradiated recipients. Peripheral blood erythrocytes (Ery), platelets (Plt), and leukocytes (leuco) were analyzed at 5, 16, and 40 weeks post-transplantation. iLT-HSCs, n = 10 recipients, donor cells from 3 pools of litters, 3 experimental days. iST-HSCs, n = 11 recipients, donor cells from 3 pools of litters, 3 experimental days. Only comparisons where p < 0.05 are displayed.

(B) Reconstitution of ABM LSK subsets with 1,000 E14.5 FL iLT-HSCs or iST-HSCs in lethally irradiated recipients after >40 weeks iLT-HSCs, n = 4 recipients, donor cells from 3 pools of litters, 3 experimental days. iST-HSCs, n = 4 recipients, donor cells from 3 pools of litters, 3 experimental days. Only comparisons where p < 0.05 are displayed.

(C) Titration of E14.5 iLT-HSCs (i) or iST-HSCs (ii) injected into lethally irradiated recipients. Plts, Ery, and leuco were analyzed after 16 weeks iLT-HSCs, n = 4–5 recipient mice per dose, donor cells from 3 pools of litters, 3 experimental days. iST-HSCs, n = 6–7 recipients per dose, donor cells from 4 pools of litters, 4 experimental days.

(D) Extreme limiting dilution analysis of LT-RU frequency (using data shown in panel D). Dotted lines represent 95% confidence interval for each population.

(E) Estimation of LT-RUs in E14.5 iLT-HSC and iST-HSC populations. n = 6 embryos from 3 litters.

(F) Secondary transplantations using 1/10^th (i) or 1/100^th (ii) doses of primary recipient bone marrow that had received a 100 cell dose of iLT-HSCs or iST-HSCs. Ery, Plts, and leuco analyzed after 16 weeks. For (i), iLT-HSC, n = 6 secondary recipients from 3 primary recipients; iST-HSC, n = 9 secondary recipients from 3 primary recipients. For (ii), iLT-HSC, n = 6 secondary recipients from 3 primary recipients; iST-HSC, n = 8 secondary recipients from 3 primary recipients. All p values shown. For (A)–(F), donor-derived reconstitution (Reconstitution %) was determined using donor-specific GFP expression. Dashed line, 1% reconstitution threshold. p values calculated from contingency analysis (Fisher’s exact test) of reconstitution outcomes.