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, 95 (23), 13726-31

Derivation of Pluripotent Stem Cells From Cultured Human Primordial Germ Cells

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Derivation of Pluripotent Stem Cells From Cultured Human Primordial Germ Cells

M J Shamblott et al. Proc Natl Acad Sci U S A.

Erratum in

  • Proc Natl Acad Sci U S A 1999 Feb 2;96(3):1162

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.

Figures

Figure 1
Figure 1
AP activity of individual human PGCs in culture. (A) Stationary and (B) migratory PGCs in a primary culture, growing on a feeder layer of mitotically inactivated mouse STO fibroblasts. (Bars represent 10 μm.)
Figure 2
Figure 2
Colony morphology. (A) Human PGC-derived cell colony growing on a feeder layer of mitotically inactivated mouse STO fibroblasts. (B) Mouse ES colony growing on a feeder layer of mitotically inactivated mouse fibroblasts. Hoffman modulation optics. (Bars represent 100 μm.)
Figure 3
Figure 3
Expression of cell surface markers by human PGC-derived cell colonies. (A) AP. (B) SSEA-1. (C) SSEA-3. (D) SSEA-4. (E) TRA-1–60. (F) TRA-1–81. (Bars represent 100 μm.)
Figure 4
Figure 4
Karyotype of human PGC-derived cell cultures. (A) XX, eight passages. (B) XY, 10 passages.
Figure 5
Figure 5
Immunohistological analysis of human EB sections. EB culture designation, antibody epitope, and objective power are as follows: (A) BF1, muscle-specific actin, ×100. Arrow indicates a cell with eccentric nuclei and cytoplasmic filaments. (B) RI5, desmin, ×60. (C) BF1, CD34, ×60. (D) BF1, S-100, ×100. (E) RI5, S-100, ×60. (F) RI, pan-neurofilament, ×100. (G) BF1, α-1-fetoprotein, ×60. (H) BF1, pan-cytokeratin, ×60. (I) RI5, cytokeratin 7, ×60. (Bars represent 20 μm.)

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