*2.6. KITD816V-TSC Show Signs of Premature Di*ff*erentiation*

In order to analyze the molecular effect of KITD816V on a cellular level in more detail, TSCs were derived from E3.5 blastocysts obtained from R26-LSL-KITD816V and Deleter-Cre mice according to published procedures [27,33]. Genotyping at passage five was used to distinguish between KITD816Vand WT-TSC lines (Supplementary Materials Figure S2A). Ultimately, we had established two lines of KITD816V-TSC which were named KITD816V #3 and KITD816V #4. In these two lines, we were able to detect a GFP-signal using fluorescence-activated cell sorting (FACS) (Supplementary Materials Figure S2B) and expression of the human *KIT* transgene using qRT-PCR (Supplementary Materials Figure S2C), demonstrating that the ROSA26-GFP-2A-KITD816 allele is functional in TSC culture in vitro. As in placental tissues, the level of endogenous murine *Kit* expression in KITD816V-TSC is not affected and remains comparable to that of WT TSC (Supplementary Materials Figure S2D).

Analysis of TSC-markers Transcription Factor AP-2 Gamma (Tfap2c), Caudal Type Homeobox 2 (Cdx2), and Eomesodermin homolog (Eomes) revealed that neither expression (Supplementary Materials Figure S2E–G) nor protein levels and distribution of TFAP2C, CDX2, and EOMES (Supplementary Materials Figure S2H) are affected in the KITD816V-TSC lines. Also, morphology as well as splitting ratio and frequency of KITD816V-TSC did not differ from WT TSC. Hence, we conclude that establishment, maintenance, and self-renewal of TSC is not altered by expression of the KITD816V transgene.

To evaluate whether KITD816V-TSC displays alterations in differentiation in vitro, WT and KITD816V lines were kept under differentiating conditions in trophoblast stem (TS) cell medium without conditioned medium (CM), Fibroblast Growth Factor (FGF) 4, and heparin for 9 days. Morphological analyses show no difference between studied lines. In both KITD816V-TSC and WT TSC, TGCs appeared in culture after three days (Figure 5A). RNA was isolated on days 0 and 6 and was analyzed for expression of P-TGC markers *Pl1*, *Pl2*, and *Plf*. Interestingly, in KITD816V-TSCs, all markers showed an increased level already at day 0, an effect which persisted to day 6 (Figure 5B). Further, spongiotrophoblast marker *Tpbpa* as well as labyrinth and S-TGC marker *Ctsq* and GlyT markers *Gjb3* and *Pcdh12* were expressed at lower levels in KITD816V TSC than in WT TSC (Supplementary Materials Figure S2I). Upregulation of *Tfap2c* which is detected during TSC differentiation was higher in KITD816V-TSC than in WT TSC [34] (Supplementary Materials Figure S2I). Of note, *Tfap2c* expression was already increased under stem cell culture conditions in KITD816V-TSC. *Hand1* is involved in mediation of TGC differentiation and is expressed in the ectoplacental cone [35]. In KITD816V-TSC it is significantly upregulated in comparison to WT-TSC after 6 days of differentiation (Supplementary Materials Figure S2I). Finally, we and others had previously shown that *Gata2* expression was upregulated upon KIT signaling in cells of the hematopoietic system [27,36]. Here, in KITD816V TSC, we detected a significant increase of *Gata2* expression after six days of differentiation whereas *Gata2* expression levels remained constant in WT TSC (Supplementary Materials Figure S2I). These results demonstrate that expression of spongiotrophoblast, labyrinth, and glycogen trophoblasts is underrepresented in KITD816V TSC upon differentiation consistent with data obtained from in vivo samples. Also, expression of KITD816V leads to an upregulation of P-TGC markers and *Tfap2c* already existing in TSC culture, which leads to premature and skewed induction of differentiation of TSCs, which is not morphologically apparent.
