Derivation and characterization of monkey embryonic stem cells

Abstract

Embryonic stem (ES) cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes. The promises and limitations of primate ES cell-based therapy are also discussed.

Figure 1

Neuronal differentiation of monkey ES cells into embryoid bodies (EBs, panel A), progenitor cells expressing nestin and musashi1 (panel B), neuronal cells expressing the neural filament TujIII (panel C) and neurotransmitter enzymes tryptophan hydroxylase (TPH, panel D), choline acetyltransferase (ChAT, panel E) and tyrosine hydroxylase (TH, panel F and G). Receptors of mature neurons such as α4-nicotinic receptors (panel H) and estrogen receptor β (panel I) were also observed. The image in panel A was taken directly from an inverted microscope. Images in panels B-H were taken after fluorescence-based immunocytochemistry. The image in panel I was taken after staining with the classic, non-fluorescent procedure using biotinylated antiserum and diaminobenzidine in the presence of nickel sulfate (shown in dark brown or black). Scale bars, when applied, are equivalent to 40 μm.

Figure 2

Neuronal differentiation of monkey ES cells under the influence of monkey brain tissue crude extracts. Images were taken before (live) and after (fixed) immunostaining for TH (green) and TujIII (red). Co-expression of TH and TujIII appears in yellow. Cell nuclei were counterstained with DAPI (blue). A and B, without extracts; C and D, with cortical extracts; E and F, with striatal extracts. Scale bars are equivalent to 50 μm.

Figure 3

Differentiation of monkey ES cells into glial phenotypes. Panel A, morphology in culture; Panel B, aggregated glial cell bundles and spheres cultured on laminin-coated glass coverslips; Panel C, expression of Schwann cell markers S100 and GFAP; Panel D, expression of Schwann cell markers GFAP and p75 (NGFR); Panel E, expression of myelin protein MBP and Schwann cell marker GC; Panel F, expression of myelin protein PLP and neuronal marker MAP2C. Panel G shows the temporal pattern of Schwann cell molecular signatures during development and a mixture of glial phenotypes derived from monkey ES cells expressing these Schwann cell markers as determined by immuocytochemistry (panel H) and RT-PCR (panel I).

Figure 4

A schematic presentation of Schwann cell markers expressed during various developmental stages. These markers are tentatively divided into 3 groups. Group 1 are genes and proteins, including p75, GFAP and SCIP (shown in blue), that express temporally during the development between immature Schwann cells and matured, myelinating cells. Group 2 markers include S100, O4 and GC (shown in red) that express during the progenitor or immature stages and continue into matured, myelinating Schwann cells. Group 3 markers include myelinating genes and proteins such as MBP and PLP (shown in green) that express only in the mature stage of myelinating Schwann cells. Monkey ES cell-derived glial phenotypes formed cell bundles and aggregates that expressed markers of all 3 groups (shown in immunofluorescence green images; nuclei were counter-stained with DAPI in blue color), except GC, which expressed in some, but not all, samples (image not shown). The insert shows gene markers detected by RT-PCR in these monkey ES cell-derived glial phenotypes.