The Cell-Surface Marker Sushi Containing Domain 2 Facilitates Establishment of Human Naive Pluripotent Stem Cells

Abstract

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.

Efficient RNA-mediated reprogramming of human somatic cells to naïve pluripotency facilitated by tankyrase inhibition

“…High efficiency with limited duration of reprogramming factor expression makes the mRNA delivery system attractive for such studies applied to primary cells. Furthermore, as illustrated in the case of XAV, it is straightforward to combine small molecules with RNA reprogramming and screen for accelerated or enhanced reprogramming, which can readily be visualised and quantified using SUSD2 live staining or flow cytometry (Bredenkamp et al, 2019).…”

“…Sushi Domain Containing 2 (SUSD2) is a cell surface protein highly expressed by human pre-implantation epiblast cells and naïve hPSC (Bredenkamp et al, 2019). By in situ live staining we detected expression of SUSD2 by the majority of compact colonies in reprogramming cultures in the presence of XAV ( Figure 1C, S1A).…”

“…Naïve hPSC were originally established and propagated in medium containing the MEK1/2 inhibitor PD0325901 (P), the glycogen synthase kinase-3 (GSK3) inhibitor CH99021 (CH), the atypical protein kinase C inhibitor Gö6938 (Gö or G) and the cytokine leukemia inhibitory factor (LIF), collectively termed t2iLGö (Guo et al, 2016;Takashima et al, 2014). More recently, however, we have found that the tankyrase inhibitor and Wnt pathway antagonist XAV939 (XAV) enhances transgene-free resetting of conventional PSC to naïve status (Bredenkamp et al, 2019;Guo et al, 2017). We therefore examined the respective effects of CH and XAV during RNA-mediated reprogramming.…”

“…Human naïve and conventional PSC are distinguished by exclusive expression of either SUSD2 or CD24 surface markers respectively (Bredenkamp et al, 2019). We quantified naïve reprogramming for HDF16, HDF75 and BJ cultures based on presence of SUSD2 and absence of CD24 after 14 days in PXGL ( Figure 2C).…”

Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

“…We demonstrate protein identification of integral membrane protein Sushi domain-containing protein 2, SUSD2 in TSC secretome in response to EVs. SUSS2 has been shown a cellsurface marker of naive pluripotency in the human embryo, with SUSD2 transcripts enriched in the pre-implantation epiblast of human blastocyst [80] and further spatially organized at the embryo-maternal interface to regulate distinct cytokine and chemokine profiles that potentially could direct trophoblast toward maternal vessels and govern local immune responses in pregnancy. [81] These results demonstrate that significant upregulated expression of SUSD2 may be transferred from endometrial EVs to reprogram the TSC environment during Table S2, Supporting Information) was investigated using KEGG pathway database.…”