Pre-Conditioning with IFN-γ and Hypoxia Enhances the Angiogenic Potential of iPSC-Derived MSC Secretome

Abstract

Induced pluripotent stem cell (iPSC) derived mesenchymal stem cells (iMSCs) represent a promising source of progenitor cells for approaches in the field of bone regeneration. Bone formation is a multi-step process in which osteogenesis and angiogenesis are both involved. Many reports show that the secretome of mesenchymal stromal stem cells (MSCs) influences the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair of the damaged area. However, the effects of iPSC-derived MSCs secretome on angiogenesis have seldom been investigated. In the present study, the angiogenic properties of IFN-γ pre-conditioned iMSC secretomes were analyzed. We detected a higher expression of the pro-angiogenic genes and proteins of iMSCs and their secretome under IFN-γ and hypoxic stimulation (IFN-H). Tube formation and wound healing assays revealed a higher angiogenic potential of HUVECs in the presence of IFN-γ conditioned iMSC secretome. Sprouting assays demonstrated that within Coll/HA scaffolds, HUVECs spheroids formed significantly more and longer sprouts in the presence of IFN-γ conditioned iMSC secretome. Through gene expression analyses, pro-angiogenic genes (FLT-1, KDR, MET, TIMP-1, HIF-1α, IL-8, and VCAM-1) in HUVECs showed a significant up-regulation and down-regulation of two anti-angiogenic genes (TIMP-4 and IGFBP-1) compared to the data obtained in the other groups. Our results demonstrate that the iMSC secretome, pre-conditioned under inflammatory and hypoxic conditions, induced the highest angiogenic properties of HUVECs. We conclude that pre-activated iMSCs enhance their efficacy and represent a suitable cell source for collagen/hydroxyapatite with angiogenic properties.

Keywords: IFN-γ; angiogenesis; hypoxia; iMSC secretome; iPSC-derived MSCs; potentiation of iMSC efficacy; pre-conditioning.

Figure 1

Timeline of IFN-γ stimulation and secretome collection: iMSCs were seeded into T-175 cell culture flasks (d0) and were stimulated with IFN-γ under hypoxic condition for 5 days (d5). Cells were washed and medium change to basal medium followed. After 24 h culture with basal medium, iMSCs secretomes were collected at day 6 (d6).

Figure 2

HLA-I and HLA-II expression of iMSCs of untreated (CO) and stimulated (with 200 ng/mL IFN-γ (IFN-γ groups) for 5 days under hypoxic and normoxic conditions. (A) Representative histograms of the surface marker expression were detected by flow cytometry. Positive cells (red), unstained control (green). (B) Amount of HLA-I and -II positive cells under the indicated conditions. Differences in surface marker expression were compared using two-way ANOVA (n = 3 patients, * p < 0.05).

Figure 3

Expression of angiogenesis-related genes by iMSCs pre-conditioned with IFN-γ under hypoxic and normoxic conditions. Gene expression levels of IL-8 (A) and VEGF-A (B) in iMSCs were quantified using a Thermo Fisher Scientific PCR instrument, and ratios of the target mRNA copy numbers related to copy numbers of the housekeeping gene (GAPDH) were calculated. Gene expressions mean values ± SEM in iMSCs from the IFN-H, IFN-N, CO-H, and CO-N groups displayed as x-fold induction values relative to the CO-N (control normoxic) group. Data were collected from three independent experiments (*** p < 0.001; **** p < 0.0001).

Figure 4

Quantification of IL-8 (A) and VEGF (B) protein concentration in secretomes from iMSCs pre-conditioned with IFN-γ under hypoxic and normoxic conditions. Values represent means ± SEM values from three independent experiments (*** p < 0.001; **** p < 0.0001).

Figure 5

Tube formation of HUVECs incubated with secretomes obtained from differently pre-conditioned iMSCs. HUVECs seeded onto the Geltrex matrix in a medium containing secretomes from differently pre-conditioned iMSCs, growth factors (+GF, positive control group), or without growth factors (−GF, negative control group). (A) Representative images (1.25× magnification, scale bar = 2000 µm) of tube formation were taken using fluorescent microscopy (calcein-AM green staining), 8 h after cell seeding. (B) Number of branches, number of meshes, and number of nodes were quantified with the ImageJ software. Values represent means ±SEM from three independent experiments (*, **, ***, **** without bar indicates significant differences to the +GF group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 6

Wound healing assay of HUVECs incubated with differently pre-conditioned iMSCs secretomes. HUVECs cultured with differently pre-conditioned iMSCs secretomes, growth factors (+GF, positive group), or without growth factors (−GF, negative control group). (A) Representative images (5× magnification, scale bar = 500 µm) of the scratched area were taken using brightfield microscopy at 0 and 8 h after cell seeding. (B) Quantification of the wound closure by HUVECs cultured with pre-conditioned iMSCs secretomes. Values represent means ± SEM from three independent experiments (* p < 0.05, ** p < 0.01).

Figure 7

Expression of angiogenesis-related genes by HUVECs cultivated in the presence of pre-conditioned iMSCs secretomes. Gene expression levels of FLT-1 (A), KDR (B), HGF (C), MET (D), MMP-1 (E), TIMP-1 (F), TIMP-4 (G), HIF-1α (H), IGFBP-1 (I), IGFBP-2 (J), IL-8 (K), and VCAM-1 (L) in HUVECs cultivated in the presence of pre-conditioned iMSCs secretomes. Quantification of mRNA levels was performed using a Thermo Fisher Scientific PCR instrument, and ratios of the target mRNA copy numbers to copy numbers of the housekeeping gene (GAPDH) were calculated in all samples. Mean values ± SEM in HUVECs from different iMSCs secretome groups are displayed as x-fold induction values relative to the negative control group (−GF). Data were collected from three independent experiments (*, **, ***, **** without bar indicates significant differences compared to the IFN-H group, # indicates that the IFN-H group show significant differences to all other groups, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Figure 8

Sprout formation by 3D HUVEC spheroids cultured within coll/HA composites in the presence of iMSC secretomes for 48 h. (A) Representative confocal images (Alexa488-Phalloidin staining) of HUVEC spheroids cultured within coll/HA composites in the presence of differently pre-conditioned iMSCs secretomes in a 10× magnification (scale bar = 200 µm). (B) Quantification of cumulative sprouts length (CSL) analyzed using the ImageJ software. Mean CSL was calculated for at least 10 randomly selected spheroids per experimental group. Values represent means ± SEM from three independent experiments (# indicates that the IFN-H HUVEC group showed significant differences to all other groups, ** p < 0.01; *** p < 0.001; **** p < 0.0001).