Tuning FOXD3 overexpression to promote specific osteogenic differentiation of human embryonic stem cells while reducing pluripotency in a 3D culture system.

Clinical use of human embryonic stem cells (hESCs) in bone regeneration applications requires that their osteogenic differentiation be highly controllable as well as time and cost effective. The main goals of the current work were thus: 1) to assess whether overexpression of pluripotency regulator Forkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in a 3D scaffolds, and 2) to evaluate if the degree of FOXD3 overexpression regulates the strength specificity of hESC osteogenic commitment. In conducting these studies, an interpenetrating hydrogel network consisting of poly (ethylene glycol) diacrylate (PEGDA) and collagen I was utilized as a 3D culture platform. Expression of osteogenic, chondrogenic, pluripotency and germ layer markers by encapsulated hESCs was measured after 2 weeks of culture in osteogenic medium in the presence or absence doxycycline-induced FOXD3 transgene expression. Towards the first goal, FOXD3 overexpression initiated 24 h prior to hESC encapsulation resulted in upregulation of osteogenic markers and enhanced calcium deposition relative to unstimulated controls, without promoting off-target effects. However, when initiation of FOXD3 overexpression was increased from 24 h to 48 h prior to encapsulation, hESC osteogenic commitment was not further enhanced and off-target effects were noted. Specifically, relative to 24 h pre-stimulation, initiation of FOXD3 overexpression 48 h prior to encapsulation yielded increased expression of pluripotency markers while simultaneously reducing mesodermal but increasing endodermal germ layer marker expression. Combined, the current results indicate that the controlled overexpression of FOXD3 warrants further investigation as a mechanism to guide enhanced hESC osteogenic commitment.