Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Abstract

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, i.e. direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, c-MYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumours in vivo after subcutaneous transplantation in SCID mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media (DM). PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene DKK3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that DKK3 transcriptionally regulated SM22 by potentiation of Wnt signalling and interaction with Kremen 1. Finally, PiPS-SMCs repopulated decellularised vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells (PiPS-ECs), leading to increased survival of SCID mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a DKK3 signalling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.