Hypoxic Preconditioning Enhances Survival and Proangiogenic Capacity of Human First Trimester Chorionic Villus-Derived Mesenchymal Stem Cells for Fetal Tissue Engineering

Abstract

Prenatal stem cell-based regenerative therapies have progressed substantially and have been demonstrated as effective treatment options for fetal diseases that were previously deemed untreatable. Due to immunoregulatory properties, self-renewal capacity, and multilineage potential, autologous human placental chorionic villus-derived mesenchymal stromal cells (CV-MSCs) are an attractive cell source for fetal regenerative therapies. However, as a general issue for MSC transplantation, the poor survival and engraftment is a major challenge of the application of MSCs. Particularly for the fetal transplantation of CV-MSCs in the naturally hypoxic fetal environment, improving the survival and engraftment of CV-MSCs is critically important. Hypoxic preconditioning (HP) is an effective priming approach to protect stem cells from ischemic damage. In this study, we developed an optimal HP protocol to enhance the survival and proangiogenic capacity of CV-MSCs for improving clinical outcomes in fetal applications. Total cell number, DNA quantification, nuclear area test, and cell viability test showed HP significantly protected CV-MSCs from ischemic damage. Flow cytometry analysis confirmed HP did not alter the immunophenotype of CV-MSCs. Caspase-3, MTS, and Western blot analysis showed HP significantly reduced the apoptosis of CV-MSCs under ischemic stimulus via the activation of the AKT signaling pathway that was related to cell survival. ELISA results showed HP significantly enhanced the secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by CV-MSCs under an ischemic stimulus. We also found that the environmental nutrition level was critical for the release of brain-derived neurotrophic factor (BDNF). The angiogenesis assay results showed HP-primed CV-MSCs could significantly enhance endothelial cell (EC) proliferation, migration, and tube formation. Consequently, HP is a promising strategy to increase the tolerance of CV-MSCs to ischemia and improve their therapeutic efficacy in fetal clinical applications.

Figure 1

The whole experimental process. CV-MSCs were treated with HP or non-HP for 24 h, then the pretreated CV-MSCs were transferred to a simulated ischemic environment for another 24 h. The survival and secretion of CV-MSC were determined. The effects of condition media obtained from CV-MSCs after ischemic stimulus on EC proliferation, migration, and differentiation were evaluated.

Figure 2

HP reduced ischemic damage of CV-MSCs. Cell number (a), total DNA (b), nuclear area (c), and cell viability (d) of CV-MSCs pretreated with HP or non-HP for 0 h, 12 h, 24 h, 36 h, or 48 h and followed by 24 h cultivation under ischemic stimulus. Data are expressed as mean ± standard deviation: ^∗p < 0.05 and ^∗∗p < 0.01 (n = 4).

Figure 3

Immunophenotype of CV-MSCs with HP and CV-MSCs with non-HP. Flow cytometry results displayed profiles of both the CV-MSCs with HP (a) and the CV-MSCs with non-HP (b) which were positive for markers CD29, CD44, CD73, CD90, and CD105, whereas they were negative for markers CD31, CD34, and CD45.

Figure 4

HP reduced the apoptosis of CV-MSCs after ischemic stimulus. Representative microphotographs taken by light microscopy (a), caspase-3 activity (b), and MTS assay (c) of CV-MSCs with HP or CV-MSCs with non-HP after ischemic stimulus for 24 h. Data are expressed as mean ± standard deviation: ^∗p < 0.05 (n = 4).

Figure 5

Effects of HP on CV-MSC biological functions after ischemic stimulus. Western blot analysis of AKT (60 kDa), p-AKT (60 kDa), and GAPDH (36 kDa) expressed in CV-MSCs with HP or CV-MSCs with non-HP (a) after ischemic stimulus for 24 h. Quantification and correlative statistical analysis (b). Data are expressed as mean ± standard deviation: ^∗p < 0.05 (n = 4).

Figure 6

Growth factor release of CV-MSCs in hypoxic condition and effect of HP on growth factor release of CV-MSCs under ischemic stimulus. Effect of hypoxic condition on growth factor release of CV-MSCs (a). Effect of HP on growth factor release of CV-MSCs cultured under ischemic stimulus for 24 h (b). Data are expressed as mean ± standard deviation: ^∗p < 0.05 (n = 4).

Figure 7

Effects of HP on CV-MSC proangiogenic properties under ischemic stimulus. After ischemic stimulus for 24 h, conditioned media collected from CV-MSCs with HP significantly enhanced HECFC survival (a), migration (b, c), and tube formation (d, e) compared to conditioned media collected from CV-MSCs without HP. The data were quantified, and statistical analyses were performed. Data are expressed as mean ± standard deviation: ^∗p < 0.05 (n = 4).