Establishment of porcine nuclear transfer-derived embryonic stem cells using induced pluripotent stem cells as donor nuclei

Abstract

We investigated whether sequential reprogramming via porcine induced pluripotent stem cells (piPSCs) or exposure to oocyte cytoplasm following nuclear transfer could generate nuclear transfer-derived ESCs (piPSCs-ntESCs). Nuclear transfer embryos were reconstructed with piPSCs possessing a ZsGreen fluorescent marker for expression of exogenous Nanog and Lin28. Reconstructed oocytes developed to morphologically normal 8-cell/morulae (35/93, 37.6%) and blastocysts (12/93, 12.9%). Although most green fluorescent protein-positive blastocysts showed efficient outgrowth (8/10, 80%), none formed primary colonies and all cultures degenerated. Conversely, 15% of fluorescent positive 8-cell/morula stage embryos showed outgrowth (6/40), with three forming primary colonies (7.5%). All three were expanded and maintained as piPSC-ntESC lines. These cell lines expressed stem cell marker genes and proteins. Despite inactivation of one X chromosome, all piPSC-ntESC lines formed teratomas comprising derivatives from all three embryonic germ layers. Strong SSEA1, 3, and 4 expression was detected at the 8-cell/morula stage in embryos reconstructed from both piPSCs and porcine embryonic fibroblasts (PEFs). SSEA3 was notably absent from IVF controls at pre-implantation embryo stages. Finally, we attempted to establish ntESCs from 8-cell/morulae reconstructed with PEFs using the same culture conditions as those for piPSC-ntESC derivation. Although eight primary colonies arose from 107 embryos (7.5%), they all degenerated after the first passage culture. Early and sustained expression of key reprogramming regulatory factors may be critical for pluripotent stem cell derivation to derive piPSC-ntESCs from 8-cell/morula stages, while the expression of SSEAs may be involved in the initial stem cell colony formation phases.

Stem cell factor’s role in enhancing the quality of fertilized and cloned porcine embryos for improved embryonic stem cell derivation

“…Finally, we investigated the effects of SCF on the implantation potential of porcine blastocysts, especially focused research on the establishment efficiency of cloned ESCs, since this model has great application value in the study of human reproductive disease via generating patient-derived blastoid or gastruloids ( 8 , 9 ), but its derivation efficiency is still low, unlike fertilized ESCs ( 39 , 40 ). Trophoblastic cells, key adhesive cells that transform into trophoblastic giant cells localized at the maternal-fetal interface, possess a spreading area that can be considered a marker of implantation potential ( 15 ).…”