Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function and they are required for vascular tissue regeneration. Multipotent adipose derived stromal cells (ASCs) can be differentiated into VSMC-like cells, which can be used as a potential VSMC source for the development of vascular tissue. However, the effects of cryopreservation on the differentiation of ASCs towards VSMCs are poorly studied to date. This study compared fresh ASCs (FA) vs. cryopreserved ASCs (CA) with respect to their differentiation potential towards VSMC-like cells. The expression of contractile VSMC markers (such as smoothelin) and cell contractility were investigated. It was found that VSMC-like cells derived from CA expressed smoothelin gene and protein at lower levels and showed compromised contractility in response to vasoconstrictors, when compared with those derived from FA. Moreover, it was demonstrated that this negative effect of cryopreservation could be mediated by MEK1/2-ERK1/2, IFNγ and Smad2/3 associated Signalling pathways. Treatment of CA with MEK1/2-ERK1/2 activator or IFNγ neutralizing antibodies enhanced Smad2/3 phosphorylation and showed a rescue of the negative effect of cryopreservation on the differentiation of ASCs towards VSMC-like cells. These findings are important for defining approaches that may use cryopreserved ASCs for vascular tissue regeneration.,Copyright ? 2018 The Authors. Published by Elsevier B.V. All rights reserved.