MSC procurement and cultivation

WJ MSCs were collected from 26 foals of full English blood mares (EBM) aged 5 to 20 yr. The umbilical cords were collected was performed following the delivery of the placenta. They were handled and processed under sterile conditions at the Tissue Engineering, Cell Therapy, and Regenerative Medicine Unit of the National Rehabilitation Institute of Mexico.

In short, a 15 to 20 cm fragment of each cord, still wrapped in amnion, was taken, followed by two washes with iodine solution interspersed with washes of sterile physiological saline solution (PSS). The sections were then cut to approximately 5 cm and stored at 4 ºC in phosphate buffered solution (PBS) with penicillin (10,000 U/ml), amphotericin B (25 µg/ml) and streptomycin (10,000 µg/ml) for processing in the laboratory. Subsequently, the WG was separated from the umbilical cord tissue and deposited in a Petri dish with PBS where cuts were made to facilitate enzymatic digestion. The latter was carried out in 10 ml of Dulbecco's modified Eagle's medium (DMEM) solution with collagenase (0.8 mg/ml) in incubation at 37 ºC for 1 hr. After the incubation time had elapsed, the cells were centrifuged at 700 xg for 7 min at 37 ºC, the supernatant was decanted and the cells were resuspended in DMEM supplemented with 10 % fetal bovine serum (FBS) and 1% penicillin, amphotericin B and streptomycin (10,000 U/ml; 25 µg/ml; 10,000 µg/ml). The primary culture was maintained in 25 cm² cell culture bottles incubated with 5 % CO2 at 37 ºC, and three passages were performed once 80 % confluence was reached.

EMF characterization

Cells obtained before the third passage were subjected to surface phenotype evaluation in order to corroborate their mesenchymal profile by flow cytometry. It was used 2.5 x 105 cells contained in round-bottom polystyrene tubes resuspended in 1ml PBS. For cell labeling, cells were incubated for 1h with specific primary antibodies for detection of CD90 (FITC Mouse Anti-Human CD90 Clone 5E10 555595), CD73 (APC Mouse Anti-Human CD73 Clone AD2560847), CD105 (PE Mouse Anti-Human CD105 Clone 266 560839), CD45 (FITC Mouse Anti-Human CD45 Clone G44-26 555478), CD34 (PE Mouse Anti-Human CD166 Clone 34 559263), CD14 (PerCP Mouse Anti-Human CD14 Clone MφP9 340585), and MHC-II (APC Mouse Anti-Human HLA-DR Clone G46-6 559866). Cells were then washed twice and analyzed using the FACS-Calibur Becton and Dickinson flow cytometer.

EMF differentiation

WJ MSCs were grown in 12-well plates at a density of 5x104 using DMEM supplemented with 5% SFB and 1% antibiotic, under the same culture conditions as previously mentioned. After 48 h, the culture medium was replaced by adipogenic, osteoblastic, and chondrogenic medium as described below.

For the adipose lineage induction, after 48 h of incubation the cell culture medium was replaced by differentiation medium formulated with DMEM supplemented with 0.5% SFB, dexamethasone (1 mM), 3-isobutylmethylxanthine (0.5 mM), insulin (10 %), indomethacin (50 mM), as well as penicillin (10,000 U/ml), amphotericin B (25 µg/ml), and streptomycin (10,000 µg/ml). Medium changes were performed every third day for 21 d. Finally, cell differentiation was evaluated using Nile red staining.

MSC differentiation into osteoblastic lineage was carried out using DMEM culture medium supplemented with SFB 1%, and with dexamethasone (100 nM), ascorbic acid (0.05 mM), 10 mM/L -glycerophosphate and BMP-7 (10 ng/ml). Likewise, the medium was changed every third day for 21 d, and osteogenic differentiation was evaluated by Von Kossa staining.

The chondrogenic lineage was obtained by using DMEM culture medium added with insulin (10 %), ascorbic acid (1 mg/ml), transforming β growth factor (TGF-β) (10 ng/ml), sodium pyruvate (1 %), and bone morphogenic protein 2 (BMP-2) (100 ng/ml). The evaluation of the differentiation was carried out with Alcian blue staining.

Horses

A total of 15 EBM horses aged 2 to 7 yr were included for this study. Twelve clinically healthy horses were used as a control group. As part of the experimental group, 3 horses with at least one episode of ERU with characteristic signs such as miosis, iris hyperpigmentation, blepharospasm, corneal edema, aqueous flame, hypopyon, hyphema, epiphora, photophobia, fibrin in the anterior chamber, conjunctival hyperemia, and scleral injection were considered.

The horses used in this study underwent a strict general physical and complete ophthalmological examination consisting of threat reflex assessment, pupillary response, consensual reflex, Schirmer's test, corneal sensibility, flourescein stain, Jones test, rose Bengal stain and fundus observation.

Tear sample collection and EMF inoculation

Once the control and experimental groups were formed, the horses were sedated using intravenous xylazine at a dose of 0.3 a 0.5 mg/kg. Subsequently, 100 µl of tears were collected using a sterile capillary tube without additives; the sample was placed in sterile vials and stored at -80 ºC until use.

Using an insulin syringe, the inoculum, PBS and 5x106 MSCs were collected for six horses of the control group, as well as for the three horses with ERU of the experimental group; both in a volume of up to 200 (l. This part of the procedure was carried out under sterile conditions. Prior to inoculum application, the area was aseptically cleaned with alcohol, avoiding direct contact with the eye. A 25 G needle was inserted into the subpalpebral area and the syringe was connected to inoculate the contents. The second and third tear samples were taken 30 min and one week post inoculation, respectively.

Evaluation of interleukins in tear samples

The evaluation of interleukins IL-1, IL-2, IL-10, IFN- and TNF in tear samples obtained before and after the treatment was performed by multiplex enzyme-linked immunoassay (ELISA), in order to determine whether there are changes in the pattern of interleukins detected.

For the multiplex ELISA test, the Equine Cytokine/Chemokine Magnetic Bead Panel Milliplex MAP Kit was used, following the manufacturer's instructions on the Luminex equipment (Bio-Plex 200, Bio-Rad Laboratories, EU). Briefly, 200 (l of wash buffer was added to each of the 96 wells of the plate, the plate was covered and incubated in agitation for 10 min at 20 ºC; the contents of the wells were then discarded, removing the excess by turning the plate over and tapping it on a bed of absorbent towels. Subsequently, 25 (l of the standard and cores were added to the corresponding wells. 25 (l of assay buffer were added to the sample wells. Subsequently, 25 (l of matrix solution were added to all wells, and 25 (l of tear sample were added to the corresponding wells. Finally, 25 (l of the bead mixture were added to each of the wells of the plate, which was incubated for 18 h under agitation at 4 ºC covered with aluminum foil.

After the incubation time had elapsed, the entire contents of the plate were discarded, and the plate was washed three times. Subsequently, 25 (l of interleukin detection antibodies were added to all wells, the plate was sealed and incubated in agitation at room temperature for 1 h. Then 25 (l of streptavidin-phycoerythrin were added to all wells, the plate was sealed and incubated in agitation at room temperature for 30 min. Once this step was completed, the contents were again decanted, and three washes were performed. To each of the wells, 150 (l of "Seath Fluid" were added and agitated for 5 min at room temperature. Finally, the plaque was read to estimate the concentration of interleukins detected in the tear samples corresponding to the three sampling times.

Statistical analysis

For the analysis of the results of the interleukin concentration, nonparametric statistics were used according to the results obtained from the homogeneity of variance (analysis of residuals) and normal distribution (Shapiro-Wilk test) tests included in the Prism 8.0 statistical program (GraphPad, Software Inc., EEUU).

In particular, the Mann Whitney U test was used to compare the concentration of cytokines between the media used (PBS and MSC) in the control group. Subsequently, this test was repeated to compare the concentration of cytokines between the control and experimental groups. Additionally, the Kruskal Wallis test followed by Dunn's multiple comparison test was performed to determine possible changes in cytokine concentration at the different times evaluated (baseline, after 30 min, and after 7 d) in both groups. In all cases, a value of P<0.05 was considered significant.

MSC procurement and cultivation

Primary cultures of MSCs obtained from WJ initially showed a rounded morphology and clustered in clusters of up to 100 m. Once attached, after 120 h they acquired fibroblastoid morphology (Figure 1).

a) Rounded morphology (white arrow) of MSCs and formation of cell clusters (black arrow); b) adherence and fibroblastoid morphology of MSCs (black arrow); c) 80 % confluence of the EMF monolayer with characteristic morphology (black arrow); c) 80 % confluence of the EMF monolayer with characteristic morphology.

Figure 1 Primary culture of mesenchymal stromal cells obtained from Wharton's jelly 

Characterization of EMF

The identification of positive MSC surface markers was carried out by flow cytometry. CD90, CD73, and CD105 markers should be expressed, while negative markers are CD14, CD34, CD45, and MHC-II. Figure 2 shows the phenotype characterization of MSCs obtained from WJ. The lack of expression of CD45, CD34, and CD14 markers is shown; so is the expression of the positive markers CD73 and CD90. On the other hand, the MScs samples evaluated showed a reduction in the expression of the CD105 marker.

Flow cytometry analysis of cultured WJ-derived MSC protein expression labeled with anti CD45 (green), CD34 (turquoise), CD14 (pink), CD73 (red), and CD90 (blue) antibodies. The histogram in purple indicates the intensity of the fluorescence of MSCs labeled with the control antibody. Open histograms indicate positive reactivity with the indicated antibody.

Figure 2 Phenotype of cultured MSCs 

MSC differentiation

MSCs were treated with three formulations of culture medium to evaluate their differentiation into adipose, osteoblastic, and chondrogenic cell lineages. Differentiation was assessed by cell staining (Figure 3); shown are representative images of a) adipocytes stained with Nile red to detect fatty acid vacuoles within cells; b) osteoblasts stained with Von Kossa stain to identify calcium deposits; c) chondrocytes detected with Alcian blue stain, mucopolysaccharide staining is visualized in the extracellular matrix of this tissue.

Figure 3 MSC differentiation to a) adipose, b) osteoblastic, and c) chondrogenic lineage 

Evaluation of horses with ERU

Horse 1. Warmblood, 12-yr-old castrated male Retinto. He presented signs of uveitis and started treatment with betamethasone, cyclosporine A, and artificial tears. Subsequently, the MSC treatment protocol was started, and only the left eye was injected; throughout the week, no clinical changes were observed.

Horse 2. Friesian, 15-yr-old whole male. He exhibited acute uveitis in the left eye, and had been previously treated with prednisolone and cyclosporine A. He showed signs such as pain, edema, vascularization, epiphora, and blepharospasm. (Figure 4). One week after the MSC treatment, he exhibited improvement from the clinical point of view; all the previous signs decreased slightly, and he showed a better mood.

a) shows blepharospasm and epiphora, b) shows edema and vascularization; palpebral margins are green due to previous fluorescein staining. c) shows a decrease in blepharospasm of the left eye d) shows decreased edema.

Figure 4 Horse 2 

Horse 3. Apaloosa, entire male, 21 yr old. He exhibited an acute condition, but received no treatment. Both eyes had miosis, epiphora, corneal edema, and neovascularization, scleral and conjunctival injection, both eyes were treated with MSCs and maintained for one week with a topical mydriatic. At 7 d both eyes were mydriatic, without pain, minor epiphora, edema, and conjunctival and scleral injection. Three days after the second visit, he started medical treatment, but he exhibited even less edema and was much more comfortable (Figure 5).

a) and b) The pictures show acute signs of uveitis of both eyes, with edema, neovascularization, and conjunctival hyperemia. c) and d) show decreased edema and hyperemia.

Figure 5 Horse 3 

Evaluation of interleukins in tear simples

Comparison between the media used (PBS and MSC) for the control group showed no differences in any of the interleukins measured. However, when comparing the concentration of these at different times (basal, after 30 min and after 7 d), it was found that the concentration of IL-1⍺ with the use of PBS as vehicle, showed statistically significant differences between the basal measurement and the one performed 7 d later (Table 1).

Since no statistically significant differences were found between the two media applied to the control group, the measurements were considered within a single group and contrasted with the data obtained in the experimental group. In this regard, no statistically significant differences were found in the concentration of ILs at any of the three times when they were used when comparing the control group with the treated experimental group.