Interrogation Of PITX3 For Blastocyst Complementation In The Generation Of Lens

Abstract

ABSTRACT Pituitary homeobox 3 (Pitx3) is a transcription factor that is involved with ocular development. Knockout of the Pitx3 gene in mice results in defects in the lens. We have developed Pitx3 knockout in porcines as a model system to assess the effects of blastocyst complements with human umbilical cord blood stem cells (hUCBSC) and human induced pluripotent stem cells (hiPSC). TALENS were used to knockout (KO) Pitx3 in porcine fibroblasts. DNA from KO fibroblasts were transferred to enucleated porcine oocytes to generate KO blastocysts. KO blastocysts and KO blastocysts with injections of hUCBSC or hiPSCs were transferred to surrogate pigs and allowed to develop to 62 days in gestation for comparisons with wild type (WT) porcine fetuses at the same gestational age. WT fetuses exhibited well-formed lenses. In contrast, the lenses in Pitx3 KO fetuses were mal-formed and poorly developed. Complementation with hiPSCs or hUCBSC did not rescue the KO lens phenotype. These results demonstrate that Pitx3 KO in porcine results in arrested lens development similar to that observed in mice. Lens complementation with hiPSC and hUCBSC, however, was not successful, possibly due to poor contribution of “non-ground state” or ‘primed’ human stem cells to the developing porcine fetus.