Figure 4.

POSTN Deficiency Leads to Expansion of Functional HSCs in FL Tissue

(A) Postn^+/+ (WT) and Postn^−/− (KO) embryos were harvested at E14.5, cleared of the extra-embryonic layers and gross morphology was compared. Scale bars, 5 mm.

(B) Individual embryos were weighed and overall weight of the WT and KO embryos was compared.

(C) Mononuclear cells from the E14.5 FL tissues were harvested and counted for the WT and KO embryos.

(D–G) The mononuclear cells harvested from the FL tissue of WT and KO embryos were analyzed for the frequency of HSC sub-populations (D) by flow cytometry. The frequency of lin⁻c-kit⁺ cells (E), LSK cells (F), and primitive HSCs (G) in E14.5 FL mononuclear cells was compared between WT and Postn^−/− embryos. n = 6–13.

(H) Schematic representation of the competitive repopulation assays. A total of 50,000 total E14.5 FL cells or 100 E14.5 FL-derived FACS-sorted SLAM LSK cells from Postn^+/+ (WT) or Postn^−/− embryos (CD45.1) was transplanted into sub-lethally irradiated Rag2^−/−γC^−/− mice (CD45.2). PB chimerism was followed for 12 weeks, after which secondary transplantations were performed using BM cells from half of the primary recipients. The secondary recipients were analyzed for PB chimerism after 12 weeks of transplantation. The PB chimerism in the rest of the primary recipients was analyzed for up to 24 weeks of transplantation. Donor-derived chimerism in BM LSK population was analyzed in moribund mice. n = 3, N = 15–18.

(I) Donor-derived PB chimerism in primary recipients analyzed after 4, 8, and 12 weeks of transplantation.

(J) After 12 weeks of transplantation of BM cells from primary recipients, secondary recipients were analyzed for PB chimerism.

(K) Multi-lineage engraftment in secondary recipients after 12 weeks of transplantation.

(L) After 24 weeks of transplantation, donor-derived PB chimerism in primary recipients that received whole FL cells from WT or KO embryos.

(M) The primary recipients analyzed after 24 weeks of transplantation for donor-derived chimerism in BM LSK population. Proportion of BM-derived cells in the total BM LSK cells was compared between the primary recipients that received whole FL cells from E14.5 WT or KO embryos (n = 3, N = 9–12).

(N) PB chimerism in primary recipients transplanted with 100 SLAM LSK cells from the E14.5 FL tissues.

(O) Secondary recipients analyzed for donor-derived PB chimerism 12 weeks after transplantation.

(P) Donor-derived multi-lineage engraftment in secondary recipients 12 weeks after transplantation.

(Q) Sorted HSCs from E14.5 WT or KO embryos were transplanted in sub-lethally irradiated animals. After 24 weeks of transplantation, donor-derived PB chimerism in primary recipients was compared between the two groups (n = 3, N = 7–11).

(R) The primary recipients analyzed after 24 weeks of transplantation for donor-derived chimerism in BM LSK population. Proportion of BM-derived cells in the total BM LSK cells was compared between the primary recipients that received freshly sorted HSCs from E14.5 WT or KO embryos (n = 3, N = 9–12).

An unpaired two-tailed Student's t test was performed. n = 3, N = 15–18, t test: ^∗p < 0.03, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.005, NS indicates not significant. See also Figure S4.