Figure 3.

SSA Enhances the Self-Renewal Capacity of HSCs Derived from Middle-Aged Mice

2,000 CD45.1 purified LSK cells from CD45.1 2-month untreated, 9-month d7shSSA, or 9-month d7SSA were transferred along with 2.5 × 10⁶ untreated 2-month CD45.2 BM cells into lethally irradiated 2-month CD45.2 primary recipients (n = 6 recipients/donor group, one individual recipient per individual donor). 12 weeks after transplant, primary recipient BM was harvested, and 2.5 × 10⁶ BM cells were transplanted along with an equal dose of untreated 2-month CD45.2 BM cells into lethally irradiated 2-month CD45.2 secondary recipients (n = 12 recipient/donor groups, two recipients per individual donor). Reconstitution of secondary recipients was measured 12 weeks after transplant.

(A) Proportion of CD45.1⁺ donor cells in BM 12 weeks following transplant in primary and secondary recipients (^∗Compared with 9-month d7shSSA mice within primary or secondary recipients. ˆCompared to untreated 2-month mice within either primary or secondary recipients. †Compared to 9-month d7shSSA between primary and secondary recipients).

(B) Proportion of donor-derived CD150⁺CD34⁻FLT3⁻ LT-HSCs and CD34⁺FLT3^hiCD62L⁺ LMPPs in the BM of secondary recipients 12 weeks after transplant.

(C) Absolute number of donor-derived LT-HSCs and LMPPs in the BM of secondary recipients 12 weeks after transplant.

(D) Concatenated flow cytometric profiles of total donor reconstitution, as well as donor B220⁺ B cells, TCRβ⁺ T cells, Gr-1⁺CD11b⁺ granulocytes, and Gr1^loCD11b⁺ in the spleen of secondary recipients 12 weeks after transplant.

(E) Absolute number of CD45.1⁺ donor cells in the spleen of secondary recipients 12 weeks after transplant.

(F) 12 weeks after secondary transplant, 2.5 × 10⁶ BM cells from secondary recipients was transplanted into tertiary recipients at a 1:1 ratio with competitor CD45.2⁺ BM, and peripheral reconstitution was assessed at 12 weeks. Concatenated FACS plots of the proportion, and the absolute number of CD45.1⁺ donor cells, in tertiary recipients (n = 6/group).

Results are expressed as mean ± SEM. ^∗/ˆp < 0.05, ^∗∗/ˆˆp < 0.01.