Abstract Study question Can alternative basement membranes support human trophoblast stem cell (hTSC) culture and recapitulate the in vivo environment more effectively than Collagen-IV (Col-IV)? Summary answer Laminin-511 (Lam-511) is the optimal alternative xeno-free matrix that enhances the stem cell state and more closely recapitulates the in vivo hTSC niche. What is known already Our knowledge on early placentation is limited due to a historical lack of robust in vitro models. Recently, hTSCs isolated from human blastocysts and first trimester placentas were successfully cultured on Col-IV, recapitulating cytotrophoblast cell self-renewal and differentiation. Even though Col-IV has been detected in the cytotrophoblast basement membrane, previously published RNA-seq analysis does not detect transcripts encoding Col-IV in the human trophectoderm. Attachment of adhesion molecules to the ECM can have an effect on cell proliferation, differentiation, polarity and gene expression. Using the correct matrix ensures a stable cell growth, as it can influence cell behaviour and identity. Study design, size, duration This was a prospective study carried out over a 6-month period. Patient-derived hTSC lines, BTS5 and CT27, obtained from RIKEN BRC Cell Bank were cultured according to the previously published protocol (1). hTSCs were maintained on culture plates coated with Col-IV, Lam-511, laminin-521 and fibronectin. Experiments were performed with biological replicates of both cell lines (n = 4), grown in every condition. Cells grown on Col-IV were used as a positive control. Participants/materials, setting, methods hTSCs were maintained on the candidate matrices for minimum of 5 passages and then were used for subsequent experiments. hTSC identity was determined by immunofluorescence analysis for known hTSC markers, GATA2 and KRT18. qRT-PCR analyses were also performed to detect additional markers EGFR, ITGA6, TEAD4, TP63, and expression from the chromosome 19 miRNA cluster (C19MC). The self-renewal capacity was assessed by cell counting and Ki67-staining. The differentiation potential was examined by performing previously published protocols. Main results and the role of chance RNA-seq analysis identified expression of ECM ligand and receptor transcripts encoding Lam-511, laminin-521 and fibronectin, which were used as candidate matrices to test. hTSC identity was maintained in all conditions as confirmed by morphological assessment, expression of known hTSC markers at the transcript and protein level, and detection of expression of C19MC miRNAs. The highest hTSC marker expression was found in cells grown on laminins. However, interestingly it was observed that only Lam-511 was capable of supporting hTSCs for more than 15 passages. Compared to Col-IV, hTSCs grown on Lam-511 appear to be more proliferative as there was a higher cell count over a 6-day period and a higher proportion of Ki-67-positive proliferative cells (p = 0.03). Additionally, preliminary data suggests that differentiation ability of hTSCs grown on Lam-511 into syncytiotrophoblasts and extravillous cytotrophoblasts is retained. Therefore, Lam-511 is an improvement to the conventional model as it enhances the stem-cell identity and closely recapitulates the in vivo niche. It can also be used as an alternative xeno-free matrix in hTSC culture in order to overcome current supply chain issues and reduce batch-to-bath variability associated with Col-IV. Limitations, reasons for caution During the 6-month period of this study, two contaminations occurred that led to slower progression of the experiments and smaller sample size. Also, there was low efficiency of the extravillous cytotrophoblast differentiation which was due to the fact that the previously published protocol performed has not yet been optimised. Wider implications of the findings Recapitulating the in vivo niche more closely will allow a deeper understanding of early placentation and help create more accurate 3D models to study the pathomechanisms of various pregnancy complications. Furthermore, utilising the xeno-free Lam-511 as an alternative matrix is a step closer to building a GMP-compliant hTSC culture. Trial registration number Not applicable
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Nov 10, 2024
Sabine Weiskirchen + 3
Just Published
Open Access