Institute for Frontier Life and Medical Sciences, Kyoto University

Self-organized formation of limb bud from pluripotent stem cells

Shunsuke Mori1,2、Eriko Sakakura1、Yuji Tsunekawa3、Masaya Hagiwara4、Takayuki Suzuki5、Mototsugu Eiraku1,6

(1 Institute for Frontier Life and Medical Sciences Kyoto University, 2 Department of Genetics, Rutgers, The State University of New Jersey, 3 Laboratory for Cell Asymmetry, Center for Biosystems Dynamics Research (BDR), RIKEN, 4 Human biomimetic system RIKEN Hakubi Research Team, 5 Laboratory of Avian Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, 6 Institute for Advanced Study of Human Biology (WPI-ASHBi), Kyoto University)

 

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

Nature Communications (2019) doi.org/10.1038/s41467-019-11702-y

Abstract

Limb development starts with formation of limb buds (LBs), which consist of tissues from different germ layers; lateral plate mesoderm (LPM)-derived mesenchyme and ectoderm-derived surface epithelium. Although various protocols for in vitro induction of three-dimensional tissues (organoids) from pluripotent stem cells (PSCs) have been reported, there has been no report of self-organized induction of trunk appendages composed of mesenchymal cells covered with epidermis such as LBs. In this study, we report a novel means for inducing a LB-like tissue from PSCs, in which a PSC aggregate spontaneously differentiates into a mesenchymal/epithelial complex tissue comprising LPM-derived limb progenitors covered with epidermis. PSC-derived LB-like tissues selectively differentiated into forelimb or hindlimb, depending on the presence or absence of retinoic acid (RA). Comparative transcriptome analysis revealed that PSC-derived LB-like tissues express a gene set closer to that of LBs than other developing mesenchymal/epithelial complex tissues such as the branchial arch, heart, and tail bud. We also found that manipulating the dorsal-ventral (D-V) polarity enabled us to artificially induce a thickened epithelial structure similar to the apical ectodermal ridge (AER) at the tip of the PSC-derived LB–like tissue. Finally, we demonstrated that the induced tissues were engrafted in an embryonic LB and adapted into digit chondrocytes and tenocytes after transplantation.
In the future, we will apply this technology to human pluripotent stem cells to establish a technique for inducing human limb bud-like tissue in vitro and explore the possibility of contributing to limb regeneration.