Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs

Jan Langkabel,Kristian Händler,Matthias Becker,Adem Saglam,Tatiana Hesse,Lisa Holsten,Yannick Riedel,Arik Horne,Anna C Aschenbrenner,Franziska Kaiser,Lorenzo Bonaguro,Kevin Baßler ,Alexej Knaus,Nico Reusch,Caroline Kubaczka,Tal Pecht,Tarek Elmzzahi,Thomas Ulas,Leon Harootoonovtch Yeghiazarian,Joachim L Schultze,Hubert Schorle

doi:10.1038/s41467-021-27586-w

Abstract

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-sequencing reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5–E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

Highlights

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments
Reprogramming of embryonic stem cells (ESCs) to iTSC was achieved by induction of transgenes encoding for Cdx[2], Tfap2c, Eomes, Gata[3], and Ets[214], and reprogramming of ESC to induced extraembryonic endoderm stem cell- (iXEN) cells is possible by overexpression of Gata[612,13,15]
We present a system for the generation of embryolike structures, termed RtL-embryoids, from a solely ESC starting population, based on combining transcription factor-mediated reprogramming paradigms and 3D co-culture

Summary

Introduction

Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. It was previously demonstrated that the three types of blastocyst-derived stem cells (ESCs, TSCs, and XEN cells) can organize into early embryo-like structures, forming tissues resembling early inner embryonic architecture, when co-cultured in 3D cell culture environments[8,9]. These structures exhibit key hallmarks of embryogenesis, such as patterning events and they were demonstrated to implant in uteri upon transplantation[9]. This two stem-cell-based system displays significantly improved developmental potential compared to previously presented ETX embryos[8]

Methods

Results

Conclusion