Directed differentiation of cES cells induced by coculture

The wide spectrum of differentiated derivatives obtained from cES cells via teratomas and EBs validates their pluripotency, but the cellular heterogeneity hinders the study of specific phenotypes. Thus we have transposed from the murine to the cynomolgus monkey system methods for haematopoietic and neural lineage-specific differentiation of ES cells, which circumvent the requirements for EB formation, addition of morphogens such as retinoic acid, or positive selection systems. Our methods depend on the coculture of cES cells with two distinct stromal cell types, PA6 and OP9, to yield relatively pure populations of either neural or haematopoietic precursors and derivatives, respectively. In this way, lineage-specific patterns of differentiation are consistently achieved.

6.1 Neural differentiation of cES cells induced by coculture with PA6 cells

We have previously described a strong neuralizing activity present on the cell surface of the PA6 line of stromal cells (originally derived from the bone marrow of mouse skull), termed stromal cell-derived inducing activity, or SDIA (1). When cocultured with PA6 cells under serum-free conditions, mES cells differentiate rapidly and efficiently into neural precursors and neurons (Figure 3): over 90% of the cells become positive for the pan-neural marker, neural cell adhesion molecule (NCAM), within a week of coculture (1, 2); and dopaminergic neurons of the midbrain type are produced at a high frequency of ~30% of induced neurons, and are transplantable (24). Our cES cells demonstrate a similar neural-differentiative capacity in the SDIA-based system (3); 45% of cells become NCAM-positive, and midbrain (tyrosine hydroxylase-positive; TH+) dopaminergic neurons are produced at a frequency of ~35% of induced neurons. (It is notable that this process of differentiation occurs within 10 d of in vitro coculture, compared with 5 weeks during cynomulgus monkey embryogenesis.) Interestingly, dorsal- and not ventral-neural markers predominate in SDIA-treated cES cells, in the absence of patterning signals. (This is in contrast to SDIA-treated mES cells, where both dorsal- and ventral-neural markers are induced.) In addition to neural tissues, SDIA-treated cES cells differentiate into eye tissues, a proportion (8%) of differentiating colonies producing patches of (Pax6+, pigmented) retinal epithelium (3, 25; and see Figure 4). Using this system, Ooto et al. (26) also reported the generation of lens tissues of characteristic transparent appearance (lentoid) in a subpopulation of differentiating cES-cell colonies.

This SDIA-based neural differentiation system has subsequently been refined (by addition of exogenous morphogens and growth factors at defined periods and concentrations, and in combinations) so that the full panoply of major

Figure 3 (see Plate 15) Immunostaining of neurons differentiated from cES cells by the SDIA method, after 13d of coculture with PA6 cells. Green fluorescence represents anti-TH staining (i.e. catecholaminergic neurons), and red, anti-TuJl staining (i.e. postmitotic neurons). Cells that are doubly positive appear yellow.

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neuroectodermal categories (dorsal and ventral) is produced by cES cells, as well as by mES cells, in vitro (24). Thus, it is inferred that in the absence of exogenous patterning signals, SDlA-treated cES cells generate naive precursors having the potential to differentiate into the full dorsal-ventral range of neuroectodermal derivatives, (24). The implications and relevance of this differentiation system for neurodegenerative diseases (such as Parkinson's disease, and retinitis pigmentosa) are highlighted.

The PA6 stromal cell line requires careful maintenance, avoiding specifically over-confluence, to preserve their neural-inducing activity. Prior to coculture, mitotic inactivation of PA6 cells may be conducted by mitomycin-C treatment (see Chapter 2, for MEF inactivation) but is not advised, as this may result in less efficient neural inducing activity.

The efficiency of neural differentiation of cES cells is affected by the quality and activity of KnockOutā„¢ SR. Therefore batch testing and titration is required to determine an optimal lot and concentration of KnockOutā„¢ SR, which may range from 5-15%. Also, inappropriate concentrations of b-mercaptoethanol cause diminished yields of neural cells.

Due to the low cloning efficiency of cES cells, partially dissociated clumps of 10-50 cells are plated onto PA6 cells, in differentiation medium. For small-scale differentiation experiments, and where cES cell cultures display spontaneous differentiation, undifferentiated colonies may be harvested manually for cocul-ture with PA6 cells. (Unusually, large cES-cell colonies tend to differentiate into non-neural derivatives, and should be avoided where possible.) For large-scale differentiation experiments it is appropriate to use enzymatic disaggregation of cES cells. After 10 d of coculture with PA6 cells, cES-cell colonies display extensive neurite projections, and are often surrounded by mesenchyme-like cells.

Figure 4 (see Plate 16) A high magnification view of retinal pigment epithelium Induced from cES cells by the SDIA method, after 24d of coculture with PA6 cells. The characteristic polygonal cells are arranged in a cobblestone fashion. These cells accumulate more pigments after an additional week of culture.

Figure 4 (see Plate 16) A high magnification view of retinal pigment epithelium Induced from cES cells by the SDIA method, after 24d of coculture with PA6 cells. The characteristic polygonal cells are arranged in a cobblestone fashion. These cells accumulate more pigments after an additional week of culture.

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