Domesticated cynomolgus monkey embryonic stem cells allow the generation of neonatal interspecies chimeric pigs

Rui Fu,Haifeng Wan,Qi Zhou,Tang Hai,Jilong Ren,Dawei Yu,Libin Wang,Wei Li,Chongyang Li,Xuepeng Wang,Ying Zhang,Tianda Li,Jing Wang,Guihai Feng

doi:10.1007/s13238-019-00676-8

Abstract

Blastocyst complementation by pluripotent stem cell (PSC) injection is believed to be the most promising method to generate xenogeneic organs. However, ethical issues prevent the study of human chimeras in the late embryonic stage of development. Primate embryonic stem cells (ESCs), which have similar pluripotency to human ESCs, are a good model for studying interspecies chimerism and organ generation. However, whether primate ESCs can be used in xenogenous grafts remains unclear. In this study, we evaluated the chimeric ability of cynomolgus monkey (Macaca fascicularis) ESCs (cmESCs) in pigs, which are excellent hosts because of their many similarities to humans. We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos, in which domesticated cmESCs (D-ESCs) injected into pig blastocysts differentiated into cells of all three germ layers. In addition, we obtained two neonatal interspecies chimeras, in which we observed tissue-specific D-ESC differentiation. Taken together, the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model, with a chimeric ratio of 0.001–0.0001 in different neonate tissues. We believe this work will facilitate future developments in xenogeneic organogenesis, bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.

Highlights

Human pluripotent stem cells have the ability to self-renew and generate a large variety of cell types, and may be capable of generating human organs in other mammals for future organ transplantation by xenogenesis (Wu et al, 2016)
We report an optimized culture medium that enhanced the anti-apoptotic ability of cynomolgus monkey (Macaca fascicularis) embryonic stem cells (ESCs) (cmESCs) and improved the development of chimeric embryos, in which domesticated cmESCs (D-ESCs) injected into pig blastocysts differentiated into cells of all three germ layers
Three criteria were used to evaluate the chimeric contributions of the cmESCs in porcine blastocysts: survival of cmESCs in the embryonic environment, the ability and efficiency of cmESCs to incorporate into total blastocysts (the number of anti-human nuclear antigen antibody-positive cells divided by the number of cells), and their ability to incorporate into the inner cell mass (ICM) (the number of anti-hNA antibody-positive cells among the Nanog homeobox (NANOG)-positive cells)

Summary

Introduction

Human pluripotent stem cells (hPSCs) have the ability to self-renew and generate a large variety of cell types, and may be capable of generating human organs in other mammals for future organ transplantation by xenogenesis (Wu et al, 2016). Primed hPSCs, which show some similarities to mouse epiblast stem cells (EpiSCs) (Nichols and Smith, 2009), can participate in normal mouse development when transplanted into gastrula-stage embryos. Region-selective hPSCs in a primed state could generate post-implantation interspecies chimeric embryos after grafting to the posterior epiblast of gastrula-stage mouse embryos (Wu et al, 2015). Primed hPSCs could not participate in normal mouse development when transplanted into preimplantation embryos as naïve PSCs (Mascetti and Pedersen, 2016), inhibiting apoptosis was shown to enhance the chimeric ability of primed hPSCs in mice (Huang et al, 2018; Wang et al, 2018b). An intermediate primed state, between the naïve and primed state, was proven to contribute to the generation of chimeras by blastocyst injection (Tsukiyama and Ohinata, 2014), and resulted in better developmental ability of a chimeric pig (Wu et al, 2017)

Methods

Results

Conclusion