Priming with caffeic acid enhances the potential and survival ability of human adipose-derived stem cells to counteract hypoxia

Abstract

The therapeutic effectiveness of stem cells after transplantation is hampered by the hypoxic milieu of chronic wounds. Prior research has established antioxidant priming as a thorough plan to improve stem cell performance. The purpose of this study was to ascertain how caffeic acid (CA) priming affected the ability of human adipose-derived stem cells (hASCs) to function under hypoxic stress. In order to study the cytoprotective properties of CA, hASCs were primed with CA in CoCl₂ hypoxic conditions. Microscopy was used to assess cell morphology, while XTT, Trypan Blue, X-gal, LDH, Live Dead, scratch wound healing, and ROS assays were used to analyze viability, senescence, cell death, proliferation, and reactive oxygen species prevalence in the cells. According to our findings, CA priming enhances hASCs' ability to survive and regenerate in a hypoxic microenvironment more effectively than untreated hASCs. Our in-vitro research suggested that pre-treatment with CA of hASCs could be a unique way to enhance their therapeutic efficacy and ability to survive in hypoxic microenvironments.

Keywords: Antioxidants; Caffeic acid; Caffeic acid, (CA); Cobalt chloride; Human adipose-derived stem cells; Human adipose-derived stem cells, (hASCs); Hypoxia stress; Mesenchymal stem cells, (MSCs); Pre-conditioning; Reactive oxygen species, (ROS); Stromal vascular fraction, (SVF); oxidative stress, (OS).

Fig. 1

Immunocytochemical expression and trilineage differentiation potential of hASCs. (a) Expression in Sec. Control. (b) Expression of CD71, CD73, CD90, and CD105. (c) Expression of CD14 and CD45 of MSCs in cultured hASCs (Magnification: 100×). (d) Trilineage differentiation potential of hASCs into adipogenic, chondrogenic, osteogenic cells. Osteogenic differentiation can be observed by Alizarin red staining of calcium phosphate produced by osteocytes; adipogenic induction can be observed by oil red O staining of lipid droplets; and chondrogenic differentiation can be observed by Alcian blue staining of proteoglycans deposition (Magnification: 200 ×, scale bar: 200 μm).

Fig. 2

Hypoxia stress optimization. (a) Microscopic analysis of diverse hypoxia doses over the morphology of hASCs (Magnification: 200× scale bar: 100 μm). (b) XTT results show the effect of hypoxia stress on cellular viability. (c) Trypan blue exclusion assay. All graphical values are represented as mean ± SD while ∗P = 0.0264, ∗∗P ≤ 0.0043, ∗∗∗P = 0.0002, ∗∗∗∗P < 0.0001 vs. Control group.

Fig. 3

Priming dose optimization. (a) Microscopic analysis of CA pre-conditioning (24 h) on the morphology of hASCs (Magnification: 200× scale bar: 100 μm). (b) XTT assay. (c) Trypan blue exclusion assay. All graphical values are represented as mean ± SD while ∗P = 0.0443 and ∗∗∗∗P < 0.0001 vs. the Control group.

Fig. 4

Efficacy of different priming doses against hypoxia stress. (a) XTT assay showing percentage viability of cells treated with different priming doses of CA under selected doses of hypoxia stress. (b) Quantitative analysis of LDH release values of cells treated with different priming doses of CA under selected doses of hypoxia stress. Values are represented as mean ± SD while ∗∗∗∗P < 0.0001 vs. Control group.

Fig. 5

Cytoprotective effect of a selected priming dose of CA against hypoxia. (a) Annexin-V immunocytochemical staining to observe apoptosis among different groups. In merged images, blue fluorescence shows staining of cells nuclei by DAPI while green fluorescence is emitted by the Annexin-V antibody stained cells (Magnification: 100×, scale bar: 105 μm) (b) Graphical representation of immunocytochemical analysis results. (c) Live/dead microscopic analysis of different experimental groups. Green and red fluorescence are emitted by live and dead cells, respectively (Magnification: 100×, scale bar: 105 μm). (d) Graphical representation of the quantitative analysis of the live/dead results. Values represent mean ± standard deviation whereas ∗P = 0.0168, ∗∗∗∗P < 0.0001vs. Control group whileP < 0.0001 vs. Hypoxia stress group.

Fig. 6

Cellular migration and senescence analysis. (a) Crystal violet stained hASCs, subjected to the scratch wound assay. Single streak images were taken at 40x magnification for all the experimental groups at a scale bar of 200 μm. (b) Graphical representation of in vitro scratch wound assay. (c) Microscopic images of cellular senescence assay, β-gal stained cells retained blue-green dye (Magnification: 100×, scale bar: 100 μm). (d) Quantitative analysis of cellular senescence assay. All graphical values are represented as mean ± standard deviation whereas ∗∗P = 0.001, ∗∗∗∗P < 0.0001 versus the Control group while ^##P = 0.001, ^###P < 0.001 and ^####P < 0.0001 versus Hypoxia stress group.

Fig. 7

ROS activity analysis. (a) DCF-fluorescence (green) images of different experimental groups of hASCs (Magnification: 100x, scale bar: 200 μm). (b) Assessment of intensity folds in different experimental groups of hASCs calculated by image J software. Data values are represented as mean ± standard deviation whereas ∗∗∗P = 0.0007 versus the Control group while ^##P = 0.007 vs. the Stress group.

Fig. 8

Gene expression analysis. (a) Pro-apoptotic markers (BAX, FADD, Casp-3). (b) Cell survival markers (IGF-1, FGF-7, Akt, PCNA, PI3–K, Bcl-xL). (c) Angiogenic markers (VEGF, SDF-1). (d) Inflammatory associated markers (IL-6, TGF-β). Data values represent mean ± standard deviation whereas ∗P ≤ 0.0246, ∗∗P ≤ 0.0061, ∗∗∗∗P < 0.0001 vs. Control group and P ≤ 0.0415, P ≤ 0.0030, ^####P < 0.0001 vs. Stress group.