Loss of p57 ^KIP2 expression confers resistance to contact inhibition in human androgenetic trophoblast stem cells

Abstract

A complete hydatidiform mole (CHM) is androgenetic in origin and characterized by enhanced trophoblastic proliferation and the absence of fetal tissue. In 15 to 20% of cases, CHMs are followed by malignant gestational trophoblastic neoplasms including choriocarcinoma. Aberrant genomic imprinting may be responsible for trophoblast hypertrophy in CHMs, but the detailed mechanisms are still elusive, partly due to the lack of suitable animal or in vitro models. We recently developed a culture system of human trophoblast stem (TS) cells. In this study, we apply this system to CHMs for a better understanding of their molecular pathology. CHM-derived TS cells, designated as TS mole cells, are morphologically similar to biparental TS (TS bip ) cells and express TS-specific markers such as GATA3, KRT7, and TFAP2C. Interestingly, TS mole cells have a growth advantage over TS bip cells only after they reach confluence. We found that p57 KIP2 , a maternally expressed gene encoding a cyclin-dependent kinase inhibitor, is strongly induced by increased cell density in TS bip cells, but not in TS mole cells. Knockout and overexpression studies suggest that loss of p57 KIP2 expression would be the major cause of the reduced sensitivity to contact inhibition in CHMs. Our findings shed light on the molecular mechanism underlying the pathogenesis of CHMs and could have broad implications in tumorigenesis beyond CHMs because silencing of p57 KIP2 is frequently observed in a variety of human tumors. complete hydatidiform mole | genomic imprinting | trophoblast stem cells | p57KIP2 | choriocarcinoma

Naive Human Embryonic Stem Cells Can Give Rise to Cells with a Trophoblast-like Transcriptome and Methylome

“…Demethylation of the H19 and KvDMR1 loci, respectively, should be expected to abrogate long-range promoter-enhancer interactions necessary for the expression of these two genes ( Soejima and Higashimoto, 2013 ). CDKN1C is of particular note because it is strongly implicated in molar pathology, and its loss eliminates contact inhibition in hTSCs ( Takahashi et al., 2019 ). However, expression of these genes was highly variable across both hTSC and tdhTSC replicates and trended lower in tdhTSCs ( Table S3 ).…”

The microRNA cluster C19MC confers differentiation potential into trophoblast lineages upon human pluripotent stem cells

“…We recently modified hTS medium to improve single-cell cloning efficiency 55 . In this study, we maintained hTS cells in the modified hTS medium, which is composed of DMEM/F12 supplemented with 1% KSR (Thermo Fisher Scientific, 10828028), 0.5% Penicillin-Streptomycin, 0.15% BSA, 1% ITS-X supplement, 200 µM L-ascorbic acid, 50 ng/ml EGF, 2 μM CHIR99021, 5 μM A83-01, 0.8 mM VPA, and 2.5 μM Y-27632.…”

“…The gRNAs were designed using the Guide design tool (Zhang Lab; no longer available) or the Custom Alt-R CRISPR-Cas9 guide RNA design tool (IDT, Coralville, IA, USA). The lentiviral vector with inducible Cas9 55 was cotransfected with pCMV-VSV-G-RSV-Rev and pCAG-HIVgp (kindly provided by Dr. H. Miyoshi, RIKEN BioResource Center, Ibaraki, Japan) into 293 T cells (Takara Bio, Kusatsu, Japan; Z2180N) using Lipofectamine LTX (Thermo Fisher Scientific, 15338030). 10 μM Forskolin was added after 24 h of transfection.…”

Regulation of human trophoblast gene expression by endogenous retroviruses

“…hTSCs were cultured according to (Okae et al, 2018), with modifications outlined in (Takahashi et al, 2019). Briefly, ~1 x 10 5 hTSC were seeded onto 6 well plates coated with either 5–10 μg/mL collagen IV (10376931, Fisher Scientific) or 0.5 μg/mL iMatrix 511 (NP892-011, Generon).…”