Abstract
Purpose:
We have demonstrated that conditioned medium (CM) obtained from developing retina can directly differentiate embryonic stem (ES) and induced pluripotent stem (iPS) cells along the retinal lineage (Parameswaran et al., 2010, 28:695-703). To examine the relative efficiency and stability of non-cell autonomous directed differentiation and to develop ES/iPS cell technology for regenerative medicine for retina, we are systematically comparing retinal CM- and small molecule-based differentiation of ES/iPS cells into retinal neurons, particularly retinal ganglion cells (RGCs).
Methods:
Neural progenitors, derived from mouse ES/iPS cells using the well-established embryoid body (EB)-based protocol (Parameswaran et al., 2010) were cultured in the presence of CM obtained from rat embryonic day 14 (E14) cells/chemically defined medium (CDM) consisting of growth factors and small molecules for 15 days. Temporal expression of retinal progenitor and RGC regulatory genes was examined to determine the recapitulation of normal mechanism of RGC differentiation. Immunohistochemical analyses were carried out to determine the acquisition of RGC phenotype. The defined medium consisted of Shh(200ng/ml), FGF8 (100ng/ml), DAPT (3uM), follistatin (100ng/ml), and cyclopamine (1ug/ml) to influence signaling pathways involved in RGC differentiation.
Results:
Both E14CM and CDM led to directed differentiation of ES/iPS cell-derived neural progenitors into RGCs. However, the recapitulation of normal developmental mechanism for and efficiency of differentiation was better with E14CM than CDM. For example, cells cultured in E14CM displayed a temporal decrease in the expression of transcripts corresponding to retinal progenitor markers (Rx and Pxa6) and a concordant increase in the expression of RGC regulators (Atoh7, Brn3b, RPF1) and mature markers (Thy1, SCNG). The concordant temporal patterns of gene expression were not detected in CDM and the levels of the expression of RGC markers were lower, as compared to those in E14CM.
Conclusions:
Our initial studies suggest that E14CM is superior to CDM in directing ES/iPS cell differentiation into RGCs.