Fig 1.
Identification of RUNX + c-Kit + HSPCs in the mouse placental labyrinth.
(A) Schematic diagrams of the mouse E12.5 placenta (top) and maternal-fetal vascular arrangement within the labyrinth (bottom) are shown. Red lines indicate fetal blood vessels. EC, fetal endothelial cells; HSPC, hematopoietic stem and progenitor cells; S-TGC, sinusoidal-trophoblast giant cells; SynT-1, syncytiocytotrophoblast layer I; SynT-2, syncytiocytotrophoblast layer II. (B) Detection of CD34 + HSPCs and ECs (green), RUNX + HSPCs (red) and c-Kit + HSPCs and trophoblasts (magenta) in wild-type E12.5 placenta (N = 3). White arrows indicate HSPCs. 1, 2 and 3 indicate boxed regions shown below. White dotted lines outline fetal blood vessels. (C) 3D snapshot view of immunostaining for c-Kit (magenta), RUNX (red), CD34 (green) and DAPI (blue) on 20 μm frozen section of E12.5 mouse placenta. White arrows indicate placenta HSPCs. (D) Immunofluorescence staining for CD34 (green), RUNX (red) and c-Kit (magenta) on wild-type E10.5 placenta sections (N = 3). The boxed region is shown at higher magnification in the panels to the right. White arrows indicate RUNX + fetal hematopoietic cells. (E) Detection of CD34 + HSPCs and ECs (green), RUNX + HSPCs (red) and c-Kit + HSPCs and trophoblasts (magenta) in wild-type E14.5 placenta (N = 3). Boxed region is shown at higher magnification in the panels on the right. No RUNX + c-Kit + HSPCs are visible. (F) Immunofluorescence staining for Ki67 (green), RUNX (red) and c-Kit (magenta) on wild-type E12.5 placenta (N = 3). Lower panels showed the boxed region in the upper left panel. White arrows indicate placenta HSPCs. (G) The percentage of Ki67 + placental RUNX + c-Kit + cells at E12.5. All scale bars = 100 μm, except 20 μm in C. The data underlying this figure can be found in S1 Data.
Fig 2.
RUNX proteins are not expressed in mouse placental ECs.
(A) Immunofluorescence staining for RUNX (red), CD34 (green) and Endomucin (magenta) in the maternal decidua of E10.5 mouse placenta sections (N = 3 placentas). Lower images indicate boxed regions 1 and 2. White arrows indicate RUNX + hematopoietic cells in maternal vessels. Yellow arrows indicate RUNX + stromal cells within the decidua. (B) Immunofluorescence staining for RUNX (red), CD34 (green) and Endomucin (magenta) in the fetal labyrinth (Lab) and chorioallantoic (CA) plate of the E10.5 mouse placenta (N = 3 placentas). Lower images show boxed regions 3 and 4. Dotted line indicates the border between the CA and labyrinth regions. White arrows indicate RUNX + stromal cells that are adjacent to Endomucin + ECs. The images in panels A and B were obtained using identical imaging settings. (C) Immunofluorescence staining for RUNX (red), CD34 (green) and Endomucin (magenta) in the E10.5 AGM region (N = 3 placentas). Lower images show boxed regions in the upper panel. White arrows indicate RUNX + hemogenic ECs. D and V indicate dorsal and ventral side of the vessel. (D) Immunofluorescence staining for RUNX (red), F4/80 (green) and Coll-I (magenta) in E10.5 mouse placenta sections (N = 3 placentas). Dotted lines outline the CA region between the umbilical cord (UC) and labyrinth (Lab). Lower images show boxed regions 5 and 6. White arrows indicate RUNX+Coll-I + stromal cells; yellow arrows indicate RUNX + F4/80 + fetal macrophages. (E) Immunofluorescence staining for RUNX (red), CD34 (green) and c-Kit (magenta) at the CA plate of the E10.5 wild-type mouse placenta (N = 3 placentas). A, artery. Scale bars: 20 μm.
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.
Fig 4.
Measurement of placental hemogenic endothelium using ex vivo HEC assays.
(A) Schematic of the OP-9 co-culture procedure used to analyze HECs capable of differentiating into blood cells ex vivo. (B) Placental labyrinth, umbilical cord (UC) and AGM were dissected from Hoxa13Cre; Ai14 E10.5 embryos (n = 6) and ECs (CD41lo/−CD45−Ter119−ESAM+CD144 + CD31 + c-Kitlo/−) fractioned into TdT + and TdT− fractions. Sorted cells (placenta TdT + /TdT− = 1,136/2000 cells; UC TdT + /TdT− = 319/1817 cells; AGM TdT + /TdT− = 0/2000 cells) were plate in limiting range in five replicates, six dilutions. (C) Graph showing frequency of HECs in each sorted population. Frequencies are shown on top of each bar. (D) Experimental design. Placental labyrinth, umbilical cord and AGM were dissected from Runx1IRES-GFP/ + E10.5 embryos (n = 30). HPC + refers to wells containing round hematopoietic cells by visual inspection. (E) Sorted GFP + cells expressing endothelial markers (CD31 + CD144 + c-Kitlo/−CD41−CD45−) from each tissue (AGM, 600 cells; UC, 153 cells; Placenta, 885 cells) were plated in OP9 co-cultures in limiting range and screened for hematopoietic outgrowth by the appearance of round hematopoietic cells after 9 days. (F) The frequency of HECs in the populations described in panel E. HEC frequencies in C and F were determined using ELDA Limiting Dilution Software. UC, umbilical cord; PL, placenta, AGM, aorta–gonad–mesonephros. The schematic diagrams in panels A and D were generated using Biorender (https://www.biorender.com/academic-license). The data underlying this figure can be found in S1 Data.
Fig 5.
Timed Cdh5-CreERT2 lineage tracing of HSPCs in the mouse placenta.
(A) Tamoxifen administration strategy for timed Cdh5-CreERT2 lineage tracing studies. Pregnant dams were orally gavaged with 4-OHT at E7.5 or E10.5 and tissues harvested at E12.5. Anticipated sites of maximal labeling (YS vs. AGM + Placenta) at each time point are indicated. (B) Representative images of Endomucin (green) and TdT (red) immunostaining of E12.5 yolk sac (YS, upper) and labyrinth from a Cdh5-CreERT2; Ai14 conceptus treated with 4-OHT at E7.5. White arrows indicate rare, TdT + placental ECs. (C) Immunofluorescence staining for TdT (red), Sox17 (green) and Endomucin (magenta) on E12.5 mouse AGM sections following Hoxa13Cre induction at E7.5 (N = 3 placentas). DA, dorsal aorta. White arrows indicate rare TdT + arterial ECs. (D) Immunofluorescence staining for TdT (red), RUNX (green) and c-Kit (magenta) on E12.5 mouse placenta sections of Ai14 control (upper panel, N = 4 placentas) and Cdh5-CreERT2; Ai14 (lower panel, N = 3 placentas) following induction with 4-OHT at E7.5. White arrows indicate RUNX + c-Kit + cells. Red arrows indicate other RUNX + hematopoietic cells. (E) Quantification of TdT + RUNX + c-Kit + cells (HSPCs) in the placenta following lineage tracing at E7.5. The absolute numbers of TdT + placental RUNX + c-Kit + cells out of the total number of placental RUNX + c-Kit + cells counted are shown. (F) Immunofluorescence staining for TdT (red), Sox17 (green) and Endomucin (magenta) on E12.5 mouse AGM sections following induction at E10.5 (N = 3 placentas). (G) Representative images of Endomucin (green) and TdT (red) immunostaining on E12.5 placenta chorioallantoic region (upper) and labyrinth (lower) from a Cdh5-CreERT2 Ai14 conceptus treated with 4-OHT at E10.5. White arrows indicate larger vessels in the chorioallantoic region (top) or placental labyrinth ECs (bottom) that were labeled. The dotted line denotes the border between the labyrinth and chorioallantoic region of the fetal placenta. (H) Immunofluorescence staining for TdT (red), RUNX (green) and c-Kit (magenta) on E12.5 mouse placenta sections of a Cdh5-CreERT2; Ai14 (N = 3 placentas) conceptus treated with 4-OHT at E10.5. White arrows indicate RUNX + c-Kit + cells in the placenta. (I) Quantification of TdT + RUNX + c-Kit + cells in the placenta following lineage tracing at E10.5. 163/231 indicates the number of TdT + cells per number of RUNX + c-Kit + cells counted. Scale bars: 100 μm (B, D and H), 50 μm (C, F and G). The data underlying this figure can be found in S1 Data.
Fig 6.
Expression of HEC marker genes in the first-trimester human placenta.
(A) Immunofluorescence staining for RUNX (red), CD31 (green) and KRT7 (magenta) on human placenta sections of 5 weeks gestational age (3 sections for N = 1 placenta). Lower panels show enlarged views of the boxed region in the upper left panel. VC, villous cord; CC, cytotrophoblast cell column. (B) Immunofluorescence staining for RUNX (red), CD31 (green) and KRT7 (magenta) on human placenta sections of 6 weeks gestational age (3 sections per placenta, N = 2 placentas). Lower panels show enlarged view of the boxed regions in the upper panels. Arrows indicate RUNX + hematopoietic cells encircled by CD31 + ECs. VC, villous cord. (C) Immunofluorescence staining for RUNX (red), CD31 (green) and KRT7 (magenta) on human placenta sections of 7 weeks gestational age (3 sections per placenta for N = 2 placentas). Lower panels show enlarged view of the boxed region in the upper panel. Arrows indicate RUNX + cells in the perivascular, stromal region of the villi. VC, villous cord. (D) Immunofluorescence staining for ALDH1A1 (red) and CD31 (green) on human placenta sections of 6 weeks gestational age (N = 2 placentas). Lower panels show enlarged view of the boxed region in the upper panel. (E) Immunofluorescence staining for ALDH1A1 (red), and CD31 (green) on human placenta sections of 12 weeks gestational age (3 sections per placenta for N = 2 placentas). Lower panels show enlarged view of the boxed regions in the upper panels. Arrow indicates abluminal ALDH1A1 + Hofbauer cells within the villi. (F) Immunofluorescence staining for KCNK17 (red), and CD31 (green) on human placenta sections of 6 weeks gestational age (3 sections per placenta for N = 2 placentas). Lower panels show enlarged view of the boxed regions in the upper panels. Scale bars, 100 μm for upper panels in A, B, C, D and upper three images in E. Twenty μm for lower panels in A, B, C, D and lower three images in E.