Lineage tracing studies suggest that the placenta is not a de novo source of hematopoietic stem cells
Fig 3
Mouse placental HSPCs are not lineage traced by Hoxa13Cre.
(A) Immunofluorescence staining for RUNX (green), TdT (red) and c-Kit (magenta) in E13.5 Ai14 only (upper) and Hoxa13Cre; Ai14 (lower) placenta (N = 3 placentas). White arrows indicate RUNX + c-Kit + cells. (B) Boxed region shown in (A) at higher magnification. (C) Quantification of TdT + placental RUNX + c-Kit + cells in Ai14 and Hoxa13Cre; Ai14 placentas (N = 3 placentas). ns, not significant. (D) Flow cytometric detection of TdT + CD34medc-Kit + cells in E12.5 placentas is shown. Red numbers indicate the percentage of TdT + cells in the gated population. (E) Quantification of percentage of TdT + cells in the CD34medc-Kit + HSPC population from the E12.5 placenta. (F) Immunofluorescence staining for c-Kit (green) and TdT (red) on E14.5 Ai14 only (upper) and Hoxa13Cre; Ai14 (lower) fetal liver sections. (G) Flow cytometric analysis of Lin−c-Kit+Sca1 + CD48−CD150 + phenotypic LT-HSCs in the E14.5 fetal liver of Hoxa13Cre; Ai14 animals. Red numbers and gates indicate percentage of TdT + cells; black numbers and gates are TdT− cells. (H) Quantification of TdT + Lin−c-Kit+Sca1 + CD48−CD150 + phenotypic LT-HSCs in the E14.5 fetal liver (FL) of Ai14 and Hoxa13Cre; Ai14 animals (n = 5 for Ai14 control and n = 3 for Hoxa13Cre; Ai14. (I) FACS plot and percentage of TdT + c-Kit + HSPCs in E14.5 fetal liver of Ai14 and Hoxa13Cre; Ai14 animals. (J) Methylcellulose colony forming unit (CFU) assays show there are no differences in the frequencies of CFUs in TdT−c-Kit + and TdT + c-Kit + progenitors (mean ± SD, n = 6). Graph on the left shows all CFUs and graph on the right are specific progenitors. GEMM, granulocyte-erythrocyte, monocyte-megakaryocyte progenitors; BFUe, burst forming unit erythroid progenitors; Mac, macrophage progenitors; GM, granulocyte-monocyte progenitors; G, granulocyte progenitors; MK, megakaryocyte progenitors. (K) Immunofluorescence staining for CD45 (green), TdT (red) and DAPI (blue) on P0 femur sections of Ai14 and Hoxa13Cre; Ai14 animals. (L) 3D Snapshot view of Hoxa13Cre; Ai14 E10.5 UCs stained for RUNX (green) and TdT (red) (N = 3 UCs). (M) Representative image showing immunostaining of E10.5 Hoxa13Cre; Ai14 umbilical cord section for TdT (red), RUNX (green) and CD34 (magenta) (three sections per cord for N = 3 umbilical cords). UA, umbilical artery; UV, umbilical vein. Lower panel shows the enlarged area in the white box from the upper panel. White arrows indicate TdT + hematopoietic cells with the hematopoietic cluster. Scale bar: 100 um. Scale bars: 100 μm (A), 50 μm (B, F, L, K and M). The data underlying this figure can be found in S1 Data.