Characterization of glial-restricted precursors from rhesus monkey embryonic stem cells

Abstract

Glial-restricted precursor (GRP) cells, the earliest glial progenitors for both astrocytes and oligodendrocytes, have been derived from embryos and embryonic stem cells (ESC) in rodents. However, knowledge regarding the equivalent cell type in primates is limited due to restrictions imposed by ethics and resources. Here we report successful derivation and characterization of primate GRP cells from rhesus monkey ESC. The purified monkey GRP cells were A₂B₅-positive and FGF2-dependent for survival and proliferation. The differentiation assays indicated that they were tri-potential in vitro and bi-potential in vivo. These newly purified GRP cells will help to facilitate understanding of the molecular mechanism of glial development in primates as well as provide a source of therapeutic donor cells for use in neuroregenerative medicine.

Keywords: Differentiation; Embryonic stem cell (ESC); Glial-restricted precursor (GRP); Rhesus monkey.

Figure 1

Direct differentiation of rhesus monkey embryonic stem cells (rESC) into early glial progenitors (GP). Undifferentiated rESC (Oct-4⁺, A) were differentiated into three distinct populations: neural rosettes (C), neuronal precursors and early GP (D–F) via embryoid body (EB). B, EB on day 5; D, phase contrast of migrating glial progenitor-like cells. Migrating bipolar cells at the periphery were stained with vimentin (E), PDGFRα (F) and A₂B₅ (arrow in F). Nuclei were stained by propidium iodide, PI (A, E) or Hoechst stain (F). Magnification: 100× (B). Scale bars: 50 μm (A, C–F).

Figure 2

Characterization of glial-restricted precursor (GRP) cells. GRP cells formed gliospheres in suspension culture (A), expressed A₂B₅/PDGFRα (B), vimentin (C) but not PSA-NCAM (D), and displayed proliferative capability (BrdU⁺, Ki-67⁺, H) under bFGF stimulation but not PDGF-AA (I). RT-PCR test showed they also expressed glial lineage transcription factor Sox9 (E). F, GRP cells had normal karyotype (42, XY) of rhesus monkey by G-band examination. G, Proliferation test showed that bFGF but not PDGF-AA was the survival factor and mitogen for GRP (n ≥ 4 in 3 independent experiments, ** represents P < 0.01). Nuclei were stained by Hoechst stain. Magnification: 100× (A). Scale bars: 50 μm (B–D, H–I).

Figure 3

Differentiation of GRP cells in vitro (A–D) and in vivo (E–H). Under appropriate differentiation conditions in vitro (Table 1), GRP cells differentiated into type II astrocytes (A₂B₅ ⁺/GFAP⁺, A), type I astrocytes (A₂B₅ ⁻/GFAP⁺, B) and oligodendrocytes (MBP⁺, C), but not neurons (MAP2⁻, D). In 2 weeks, transplanted green fluorescent protein, GFP (E, F) or PHK-26 labeled (G, H) GRP cells migrated and integrated into host Sprague Dawley (SD) rats, differentiating into oligodendrocytes (MBP⁺, F, G) and astrocytes (GFAP⁺, E). Neurons were not detected (MAP2⁻, H). Nuclei were stained by Hoechst stain. Scale bar: 100 μm.