Small Molecule Inhibitor Neural Differentiation of Marmoset Monkey iPS cells

The technology to derive induced pluripotent stem (iPS) cells in a patient-specific manner holds promise for autologous regenerative cell therapies.  In order to develop iPS-derived cranial-specified cells for orofacial reconstruction, the generation of cranial neural crest cells in vitro is highly desirable.  The development of a non-human primate iPS cell model is beneficial for future assessment of autologous cell therapy safety and efficacy.  Recently, our lab developed iPS cells from the common marmoset (Callithrix jacchus), a non-human primate widely employed in biomedical research.  Objective: To determine the feasibility of using small molecule inhibitors for neural and neural crest differentiation of marmsoset monkey iPS cell in vitro.  Methods:  Marmoset iPS cells were allowed to form small embryoid bodies under non-adherent conditions for 5 days in the presence of TGF-β/Activin inhibitor (SB431542), BMP inhibitor (dorsomorphin), and ROCK/Rho inhibitor (Y27632) in neural medium, followed by adherent culture for 1 day on Matrigel.  Indicators of neural differentiation (SOX1) and neural crest differentiation (SNAI2, SOX10, and TFAP2B) were assessed by qRT-PCR.  Results: Day 6 treated marmoset cells yielded a 74-fold increase of gene expression of the early neuroectoderm marker, SOX1; 79-fold increase in the neural crest-mesoderm marker, SNAI2; 18-fold increase in the neural crest marker, SOX10, and a 6-fold increase in the neural crest marker TFAP2B.  Treated cells show a distinct cell morphology different from undifferentiated marmoset iPS cells.  Conclusions: We demonstrate that combinatory treatment of small molecule inhibitors in an embryoid body formation protocol can effectively differentiate marmoset iPS cells in vitro into the neural lineage, including upregulation of neural crest markers SNAI2, SOX10, and TFAP2B.  Utilization of this strategy for neural differentiation allows for avoidance of stromal factor-derived methods that rely on co-culture with mouse cell lines such as PA6 or MS5.  This work was supported by NIDCR, Grant #DE14318 for the COSTAR Program.