Figure 5. Expression of Ucn 3 in hESC-cell derived alpha and beta cells requires in vivo maturation.

Quantitative PCR analysis of key differentiation stages towards the human beta cell lineage reveals no Ucn 3 expression at the embryonic stem cell (ES) or definitive endoderm (DE) stage. While pancreatic cultures enriched for pancreatic endoderm cells (PE) by CD142 did not express Ucn 3, polyhormonal cells (PH) enriched from the same cultures by CD200, started to express Ucn 3. Implants of hESC-derived pancreatic progenitors robustly express Ucn 3 after differentiation and maturation in vivo at 140 days post-transplant (Tx). Ucn 3 expression is measured by qRT-PCR, normalized to TATA-box binding protein (TBP) and plotted relative to ES cells (A). These observations are confirmed on in vitro differentiated hESC-derived pancreatic endoderm, which contains a minor fraction of polyhormonal cells that express insulin (B) and glucagon (C), but are mostly devoid of Ucn 3 immunoreactivity with the exception of faint Ucn 3 expression in the occasional glucagon⁺ cell (C, inset). Ucn 3 immunoreactivity is robustly upregulated 18 weeks following engraftment in the epidydimal fat pads of mice and demonstrates significant Ucn 3 colocalization with insulin⁺ beta cells and glucagon⁺ alpha cells (D). Ucn 3 immunoreactivity is robustly upregulated 140 days post implantation in TheraCyte encapsulation devices and co-localizes with both insulin (E) and glucagon (F). Heterogeneity exists among both alpha and beta cells regarding the expression of Ucn 3. Arrows indicate Ucn 3⁺ cells that co-express insulin (G) or glucagon (H).