Self-organized formation of developing appendages from murine pluripotent stem cells

Shunsuke Mori,Yuji Tsunekawa,Masaya Hagiwara,Takayuki Suzuki,Mototsugu Eiraku,Eriko Sakakura

doi:10.1038/s41467-019-11702-y

Abstract

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectoderm-derived surface epithelium. Here, we report means for induction of an LB-like mesenchymal/epithelial complex tissues from murine pluripotent stem cells (PSCs) in vitro. The LB-like tissues selectively differentiate into forelimb- or hindlimb-type mesenchymes, depending on a concentration of retinoic acid. Comparative transcriptome analysis reveals that the LB-like tissues show similar gene expression pattern to that seen in LBs. We also show that manipulating BMP signaling enables us to induce a thickened epithelial structure similar to the apical ectodermal ridge. Finally, we demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation. This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs.

Highlights

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectodermderived surface epithelium
We demonstrate that the surface ectoderm in mouse embryonic stem cells (mESCs)-derived LBs (ES–LBs) forms an AERES-like structure in response to an artificially manipulated bone morphogenetic protein (BMP) signaling, and ES–LBs were functionally integrated after transplantation into the developing mouse limbs
Almost all mESC aggregates treated with BMP4 in the presence of Matrigel that we observed had differentiated into posterior primitive streak (PPS) (7Tcf+) and into the lateral plate mesoderm (LPM) (Hand2+)

Summary

Introduction

Limb development starts with the formation of limb buds (LBs), which consist of tissues from two different germ layers; the lateral plate mesoderm-derived mesenchyme and ectodermderived surface epithelium. We demonstrate that the induced tissues can contribute to endogenous digit tissue after transplantation This PSC technology offers a first step for creating an artificial limb bud in culture and might open the door to inducing other mesenchymal/epithelial complex tissues from PSCs. A three-dimensional (3D) culture of PSCs has been reported to enable the formation of organoids with various well-recapitulated aspects of organogenesis, such as tissue patterning, morphogenesis, proper arrangement of each cell type, and developmental timing[1]. At a later stage of development, LBs grow in the distal direction and form orderly patterned digits Both LB outgrowth and digit patterning require the formation of two signaling centers in LB; the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA). We demonstrate that the surface ectoderm in mESC-derived LBs (ES–LBs) forms an AERES-like structure in response to an artificially manipulated BMP signaling, and ES–LBs were functionally integrated after transplantation into the developing mouse limbs

Methods

Results

Conclusion