Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming

Abstract

Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was > 4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.

Zika virus impacts extracellular vesicle composition and cellular gene expression in macaque early gestation trophoblasts

“…There were no differences in CG or progesterone secretion between infected and control samples for TSC, ST3D, or EVTs (Supplemental Figure 4A). TSCs secreted minimal CG or progesterone and ST3Ds and EVTs secreted signi cantly more CG than TSCs, in agreement with our previous report 12 .…”

“…The placenta is comprised of several trophoblast cell types, including villous cytotrophoblasts (vCTBs), syncytiotrophoblast (STs), extravillous trophoblasts (EVTs), as well as fetal macrophages (Hofbauer cells), other immune and vascular cells, and broblasts 10 . vCTBs can be programmed to a proliferative stem cell-like state in vitro, termed trophoblast stem cells (TSCs) that can be differentiated into STs or EVTs [10][11][12] .…”

“…In the current study, a rhesus macaque TSC model was utilized as it allows for the study of trophoblast-type responses. Macaque TSCs derived from rst trimester vCTB maintain cellular proliferation and can be directed to ST-or EVT-speci c differentiation 12 . We previously showed that TSCs differentiated to ST display features characteristic of early rst-trimester, a developmentally critical period before the de nitive placenta has completely formed.…”

Transcriptomic analysis of primate placentas and novel rhesus trophoblast cell lines informs investigations of human placentation

“…Because the placenta is a heterogeneous organ comprised of many cell types in addition to trophoblasts, such as immune, stromal, and vascular cells [ 46 ], we next sought to isolate primary trophoblasts from bulk rhesus placentas for immortalization and characterization, including a comparison of gene expression. While previous studies have successfully isolated and cultured primary trophoblasts from first and third trimester rhesus placenta [ 47 – 50 ], as well as generated rhesus blastocyst- and placenta-derived trophoblast stem cells [ 51 , 52 ], no rhesus immortalized trophoblast cell lines currently exist for in vitro investigations. Using the strategy described in Fig.…”

“…Since the immortalized trophoblast cell lines were generated from rhesus placental tissue near the peak of mCG secretion, and both hCG and mCG are predominantly secreted from the STB during pregnancy, we interpreted the lack of mCG secretion by the immortalized rhesus trophoblast lines as further support for an EVT-like phenotype. It should be noted that there are previous reports of CG expression in human EVTs [ 98 – 100 ], and while a similar rhesus study has not been conducted in vivo, the recent derivation of rhesus trophoblast stem cells indicates that EVT-like cells do secrete CG in vitro [ 52 ]. Nonetheless, iRP-D26 and iRP-D28A showed overall transcriptomic similarity to primary rhesus trophoblasts and retained key expression and functional characteristics of invasive EVTs, highlighting the suitability of these lines for future in vitro functional investigations.…”