Figure 2. Epcam^hi cells represent multipotent labyrinth trophoblast progenitors.

(A) Schematic for analysis of the developmental potential of Epcam^hi cells in vitro. Epcam^hi cells from E10.5 placenta were enriched using magnetic beads and co-cultured with OP9 for 7 days. QRT-PCR documents the expression of Itga4 in Epcam^hi cells and Vcam1 in OP9.

(B) QRT-PCR showing the maintenance of SynT specific genes and up-regulation of markers for differentiated SynT-I and -II and sTGC in Epcam^hi cultures after 7 days.

(C) Documentation of increased frequency of Cdh1⁺ SynT with multiple nuclei after 7 days in culture.

(D) Documentation that Cdh1⁺ multinucleated cells co-express trophoblast marker CD9.

(E) Schematic for in vivo clonality analysis. Rosa26 (R26) Rainbow reporter mice were mated with R26 CreER^T2 mice. At E9.5, Cre-mediated gene deletion was induced by injection with 4OH-tamoxifen, and embryos were dissected at E12.5.

(F) Fluorescence image indicating that Cre-mediated gene recombination induces multi-color labeling and establishment of clones of labeled cells in the labyrinth. JZ, junctional zone. La, labyrinth. Me, mesenchyme.

(G) Documentation of multi-lineage differentiation in a cluster of mCherry⁺ labeled trophoblasts. (i) Epcam^hi (LaTP), (ii) SynT (Epcam^low/neg) and (iii) sTGC (Epcam^neg with large nuclei).

(H) Analysis of the frequency of clusters containing mCherry-labeled Epcam^hi cells documenting infrequent labeling of clusters with same fluorescent color.

(I) Average number of mCherry⁺ cells in a labeled cluster documenting the establishment of multicellular, labeled clones.

(J) Differentiation potential of mCherry-labeled cells documenting the presence of clones with Epcam^hi LaTP and all labyrinth trophoblast subtypes.

(K) Schematic representing the hierarchy of trophoblast-lineage differentiation. LaTP gives rise to all types of labyrinth trophoblasts, but not Sp or TGC.

All error bars indicate SEM (Standard error of mean).

See also Figure S2.