Derivation of pluripotent stem cells from cultured human primordial germ cells

TLDR

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. The study cultured gonadal ridges and mesenteries containing primordial germ cells on mouse STO feeder layers with leukemia inhibitory factor, basic fibroblast growth factor, and forskolin to establish pluripotent stem cell lines. Single PGCs were visualized by alkaline phosphatase staining and, during culture, most colony cells remained alkaline phosphatase‑positive and expressed the stem cell markers SSEA‑1, SSEA‑3, SSEA‑4, TRA‑1‑60, and TRA‑1‑81. Over 7–21 days, the PGCs formed large, karyotypically normal colonies resembling embryonic stem and embryonic germ cells that differentiated into all three germ layers and were obtained as both XX and XY cultures, meeting criteria for pluripotent stem cells.

Abstract

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5–9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7–21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.