Tuning Forkhead Box D3 overexpression to promote specific osteogenic differentiation of human embryonic stem cells while reducing pluripotency in a three ‐dimensional culture system

AbstractClinical 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 (a) to assess whether overexpression of pluripotency regulatorForkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in three ‐dimensional (3D) scaffolds and (b) to evaluate if the degree ofFOXD3 overexpression regulates the strength and specificity of hESC osteogenic commitment. In conducting these studies, an interpenetrating hydrogel network consisting of poly(ethylene glycol) diacrylate 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‐inducedFOXD3 transgene expression. Towards the first goal,FOXD3 overexpression initiated 24  hr prior to hESC encapsulation, relative to unstimulated controls, resulted in upregulation of osteogenic markers and enhanced calcium deposition, without promoting off‐target effects. However, when initiation ofFOXD3 overexpression was increased from 24 to 48  hr prior to encapsulation, hESC osteogenic commitment was not further enhanced and off‐target effects were noted. Specifically, relative to 24‐hr prestimulation, initiation ofFOXD3 overexpression 48  hr prior to ...
Source: Journal of Tissue Engineering and Regenerative Medicine - Category: Biotechnology Authors: Tags: RESEARCH ARTICLE Source Type: research