Differentiation of nonhuman primate embryonic stem cells along neural lineages

Abstract

The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14-16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), beta-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and beta-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, beta-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen beta-tublinIII (73+/-4.7%) and some expressed intermediate neuron antigen MAP2 (18+/-7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%+/-1.2%), GFAP (4.93+/-1.9%), S100 (7+/-3.5%) and O4 (0.27+/-0.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.

Fig. 1

The protocol for ORMES6 ESC differentiation into neural lineage cells. (A) Sequential culture procedures for ORMES6 ESC differentiation into neural lineage subtype cells. (B) ORMES6 ESC colony (arrow) in an undifferentiated state on an MEF feeder layer; (C) EBs derived from ORMES6 ESCs; (D) columnar rosette cells (arrow) resembling the early neural tube in the EB outgrowth cells after exposure to bFGF for 14–16 days; (E) isolated rosette cells formed free-floating cell aggregates similar to neurospheres; (F) highly branched cells, similar in morphology to neural lineage cells, grew out from attached rosette cell aggregates. Neurite-like processes appeared after removing bFGF from the cell culture medium; (G) fibroblastoid and epithelia-like cells that result from the spontaneous differentiation of ORMES6 cells in the absence of cytokines and chemical inducers. Scale bar: 40 μm.

Fig. 2

Undifferentiated ORMES6 ESCs express embryonic cell markers. (A) SSEA 4 (green); (B) Oct4 (red); (C) glycoprotein TRA-1-60 (green)/ Oct4 (red) and (D) glycoprotein TRA-1-81 (green)/Oct4 (red). The nuclei were stained with DAPI (blue). The undifferentiated ORMES6 ESCs expressed SSEA 4, Oct4, TRA-1-60 and TRA-1-81 antigens. Cells only stained with DAPI were MEF feeder cells. Scale bar: 40 μm.

Fig. 3

RT-PCR analysis of gene expression at select stages of differentiation. Oct4, Sox2 and NANOG are markers of pluripotent stem cells. MAP2, β-tublinIII, NeuroD are neuronal markers, and GFAP is glial cell marker. Undifferentiated ORMES6 ESCs expressed Oct4, NANOG, Sox2 stemness genes and MAP2, β-tublinIII neuronal genes. Oct4 and NANOG transcription factors were markedly down-regulated in the rosette cells. Sox2, MAP2, β-tublinIII neuronal genes and GFAP glial cell genes were expressed in the rosette cells. The neural subtype cells derived from rosette cells expressed Sox2, MAP2, β-tublinIII, GFAP and NeuroD genes. However, spontaneously differentiated cells only expressed β-tublinIII, Sox2 and MAP2 genes. Lane 1: undifferentiated ORMES6 ESCs; Lane 2: neural progenitor cells (neural tube like-rosette cells) induced by neural induction medium (Step 2 of protocol); Lane 3: Neural subtype cells derived from rosette cells (Step 4); Lane 4: Spontaneously differentiated ORMES6 ESCs cultured in ESC medium without MEF cells.

Fig. 4

EBs derived from ORMES6 ESCs express markers of three germ layers. EBs derived from ORMES6 ES cell line, cultured with ES cell culture medium in low-attachment 6-well plates for 1 month, expressed three germ layer markers. (A) β-tublinIII as an ectodermal-derived neuronal marker (red label); (B) alpha-fetoprotein as an endodermal marker (green label); (C) cardiac troponin I as a mesodermal marker (green label) and (D) brachyury as a mesodermal marker (green label). The nuclei were counter-stained with DAPI (blue). Scale bar: 40 μm.

Fig. 5

Expression of neuroepithelial markers by neural tube-like rosette cells. After exposure to bFGF for 14–16 days, neural tube-like rosette cells formed in the EBs outgrowth. The isolated rosette cells were positively stained for nestin, FoxD3, Pax6 and Sox10 that are considered as markers for neuroepithelial stem cells. The neural rosette cells were also positive for Ki67, which is a cell proliferation marker. (A) Nestin (green label); (B) FoxD3 (red label); (C) Pax6 (green label); (D) Sox10 (green label); (E) Ki67 (red label) and (F) percentage of each marker. The nuclei were counter-stained with DAPI (blue). Scale bar: 40 μm.

Fig. 6

Neural subtype cells derived from the rosette cells. The neural subtype cells differentiation was initiated by removal of bFGF from the culture medium. Most differentiated cells expressed neuronal cell markers, a small subpopulation of the differentiated cells expressed glial lineage cells markers. (A) Neuronal marker β-tublinIII (red label); (B) neuronal-specific protein MAP2 (red label); (C) glial cell marker GFAP (red label); (D) glial cell marker S100 (red label); (E) glial cell marker A2B5 (green label) and (F) oligodendrocyte marker O4 (green label). The nuclei are counter-stained with DAPI (blue). Scale bar: 40 μm. (G) The percentages of markers expressed by the differentiated neural subtype cells.

Fig. 7

Neural rosette cells derived from ORMES6 gave rise to neuron and glial cells in vivo. The rosette cells were transplanted into the striatum of NIHIII mice. At 1, 2, 4 weeks after transplantation, the mice were sacrificed and the brains collected for analysis. The cells could be found at 1 week (n = 3) and 2 weeks (n = 3) post-transplantation. And the number of surviving cells is very small. By 4 weeks, cells were not detected (n = 3). The transplanted cells were distinguished from the host cells by human-specific nuclear antigen (HNA) staining. The transplanted neural rosette cells expressed cell proliferation marker Ki67, astrocyte marker GFAP and neuron markers MAP2 and NeuN. (A) DAPI (blue label); (B) HNA (green label); (C) cell proliferation marker Ki67 (red label); (D) Merge; (E) DAPI (blue label); (F) HNA (green label); (G) neuronal-specific protein MAP (red label); (H) Merge; (I) DAPI (blue label); (J) HNA (green label); (K) neuron marker NeuN (red label); (L) Merge; (M) DAPI (blue label); (N) HNA (green label); (O) astrocyte marker GFAP (red label); and (P) Merge. The nuclei are counter-stained with DAPI (blue). Scale bar: 50 μm.