Equine Hoof Progenitor Cells Display Increased Mitochondrial Metabolism and Adaptive Potential to a Highly Pro-Inflammatory Microenvironment

Abstract

Medicinal signaling cells (MSC) exhibit distinct molecular signatures and biological abilities, depending on the type of tissue they originate from. Recently, we isolated and described a new population of stem cells residing in the coronary corium, equine hoof progenitor cells (HPCs), which could be a new promising cell pool for the treatment of laminitis. Therefore, this study aimed to compare native populations of HPCs to well-established adipose-derived stem cells (ASCs) in standard culture conditions and in a pro-inflammatory milieu to mimic a laminitis condition. ASCs and HPCs were either cultured in standard conditions or subjected to priming with a cytokines cocktail mixture. The cells were harvested and analyzed for expression of key markers for phenotype, mitochondrial metabolism, oxidative stress, apoptosis, and immunomodulation using RT-qPCR. The morphology and migration were assessed based on fluorescent staining. Microcapillary cytometry analyses were performed to assess the distribution in the cell cycle, mitochondrial membrane potential, and oxidative stress. Native HPCs exhibited a similar morphology to ASCs, but a different phenotype. The HPCs possessed lower migration capacity and distinct distribution across cell cycle phases. Native HPCs were characterized by different mitochondrial dynamics and oxidative stress levels. Under standard culture conditions, HPCs displayed different expression patterns of apoptotic and immunomodulatory markers than ASCs, as well as distinct miRNA expression. Interestingly, after priming with the cytokines cocktail mixture, HPCs exhibited different mitochondrial dynamics than ASCs; however, the apoptosis and immunomodulatory marker expression was similar in both populations. Native ASCs and HPCs exhibited different baseline expressions of markers involved in mitochondrial dynamics, the oxidative stress response, apoptosis and inflammation. When exposed to a pro-inflammatory microenvironment, ASCs and HPCs differed in the expression of mitochondrial condition markers and chosen miRNAs.

Keywords: ASC; adipose stem cells; equine; hoof; laminitis; progenitor cells; stem cells.

Figure 1

Characterization of ASC and HPC morphology and gene expression of surface markers. (A) Representative confocal photographs of ASCs and HPCs labelled with DAPI (blue), phalloidin (green) and MitoRed (red) showcase differences in cellular morphology. (B) The phenotypes of ASC and HPC native populations were assessed using RT-qPCR to determine the relative expression analysis of surface markers (CD105, Nestin, CD29), angiogenesis markers (Ang1, Igf1, Hif1a, Vegfa) and matrix metalloproteinases (Mmp2, Mmp9, Mmp14). Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 2

(A) Scratch wound healing assay of ASC and HPC native populations. Scale bar = 200 µm. (B) Pararosaline staining of the scratch wound healing assay performed 48 h after the scratch wound. (C) Analysis of colony-forming efficiency in ASC and HPC native populations. (D) The distribution of ASCs and HPCs in the cell cycle (blue-G0/G1; pink-S; olive-G2/M, green-ungated/debris). Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 3

(A) Mitochondrial membrane polarization analysis of HPC and ASC native populations. The analysis was performed with the Muse^® MitoPotential Kit. (B) Gene expression of key mitochondrial dynamics markers (Fis1, Mfn1, Dnm1l, Pink1, Mief2, Mief1, Opa1, Rhot1). The mitochondrial dynamics in HPC and ASC native populations were tested using a RT-qPCR assay. Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 4

(A) Gene expression of key oxidative stress markers (Sod1, Sod2, Cat1) assessed using RT-PCR. (B) Characterization of oxidative stress in HPC and ASC native populations. The analysis was performed using the Muse^® Oxidative Stress Kit. (C) Nitric oxide activity analysis in ASC and HPC native populations. The analysis was performed using the Muse^® Nitric Oxide Kit. Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 5

Gene expression of apoptosis markers (p21, p53, Bax, Bcl2, Casp3, Casp9) and immunomodulatory mediators (Il6, Il8, Il10, Il13, Il1b, Tnfa, Tgfb1, Mcp1, Nfkb, Nfkbia, Ikbkb) in native ASCs and HPCs, assessed using RT-qPCR. Results are expressed as mean  ±  SD.Statistically significant differences are marked with an asterisk (** p  <  0.01, *** p  <  0.001).

Figure 6

(A) Gene expression of key mitochondrial dynamics markers (Fis1, Mfn1, Pink1, Dnm1l, Opa1, Rhot1) in control (CTRL) and cytokine cocktail-treated (CC) ASCs and HPCs, assessed with RT-qPCR. (B) Confocal photographs of the control (CTRL) and cytokine cocktail-treated (CC) ASCs and HPCs labelled with DAPI (blue) and MitoRed (red). (C) Gene expression of key mitochondrial metabolism markers (Mrpl24, Mterf4, Ppargc1b, Uqcrc2, Oxa1l, Coxhi1, Ndufa9, Pusl1, Mief1, Mief2) in control (CTRL) and cytokine cocktail-treated (CC) ASCs and HPCs, assessed using RT-qPCR. Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 7

Gene expression of (A) apoptosis markers (p21, p53, Casp9, Bax, Bcl2), (B) immunomodulatory mediators (Il6, Il8, Il10, Il13, Il1β, Tgfβ) and (C) oxidative stress markers (Sod1, Sod2, Cat1) in control (CTRL) and cytokine cocktail-treated (CC) ASCs and HPCs, assessed with RT-qPCR. Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 8

The expression of miRNAs (miR-21-5p, miR-27a, miR-30c-5p, miR-34a-5p, miR34c, miR-96-5p, miR-125a, miR-125b-5p, miR-218, miR-451) in (A) native ASCs and HPCs, (B) cytokine cocktail-treated (CC) ASCs and HPCs, assessed using RT-qPCR. Results are expressed as mean  ±  SD. Statistically significant differences are marked with an asterisk (* p  <  0.05, ** p  <  0.01, *** p  <  0.001).

Figure 9

Graphical representation of the study design. P1 (Proximal phalanx bone), P2 (Middle phalanx bone), P3 (Distal phalanx/coffin bone).