TuningForkhead Box D3overexpression to promote specific osteogenic differentiation of human embryonic stem cells while reducing pluripotency in a three‐dimensional culture system

Abstract

Clinical use of human embryonic stem cells (hESCs) in bone regeneration applications requires that their osteogenic differentiation be highly controllable as well as timeand cost-effective. The main goals of the current work were thus (a) to assess whether overexpression of pluripotency regulator Forkhead Box D3 (FOXD3) can enhance the osteogenic commitment of hESCs seeded in three-dimensional (3D) scaffolds and (b) to evaluate if the degree of FOXD3 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-induced FOXD3 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 of FOXD3overexpression 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 of FOXD3 overexpression 48 hr prior to encapsulation yielded increased expression of pluripotency markers while 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.

FOXD1 Repression Potentiates Radiation Effectiveness by Downregulating G3BP2 Expression and Promoting the Activation of TXNIP-Related Pathways in Oral Cancer

“…In addition, FOX transcription factors have been shown to associate with cancer research such as drug resistance, tumor growth, genomic alterations or drivers of initiation and thought to be a potential therapeutic strategy to combat cancer and putative biomarkers for specific cancers [ 6 , 10 ]. The FOXD subfamily includes FOXD1 , FOXD2 , FOXD3 and FOXD4 and functions as a critical regulator in normal cell development and disease progression [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. Moreover, the FOXD subfamily has also been found to play a role as an important regulator for tumorigenesis and cancer progression [ 19 , 20 , 21 , 22 , 23 , 24 ], e.g., therapeutic resistance and cancer metastasis, and serves as a prognostic biomarker in several types of cancer [ 25 , 26 , 27 ].…”

The Role of Forkhead Box Family in Bone Metabolism and Diseases

“…Huang et al pointed out that FoxD3 may mediate transcriptional activation of miR-496 triggered by IL-1β, thereby repressing the Wnt/ β-catenin signaling pathway and reducing osteoblast differentiation (Huang and Chen, 2017). Interestingly, a study by Kamaldinov et al showed that overexpressed FoxD3 may enhance osteogenesis in human embryonic stem cells via the endochondral ossification pathway (Kamaldinov et al, 2018).…”