Use of adipose-derived mesenchymal stem cells in keratoconjunctivitis sicca in a canine model

Abstract

Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is an immune-mediated multifactorial disease, with high level of prevalence in humans and dogs. Our aim in this study was to investigate the therapeutic effects of allogeneic adipose-derived mesenchymal stromal cells (Ad-MSCs) implanted around the lacrimal glands in 12 dogs (24 eyes) with KCS, which is refractory to current available treatments. Schirmer tear test (STT) and ocular surface integrity were assessed at 0 (before treatment), 3, 6, and 9 months after treatment. Average STT values and all clinical signs showed a statistically significant change (P < 0.001) during the follow-up with reduction in all ocular parameters scored: ocular discharge, conjunctival hyperaemia, and corneal changes, and there were no signs of regression or worsening. Implanted cells were well tolerated and were effective reducing clinical signs of KCS with a sustained effect during the study period. None of the animals showed systemic or local complications during the study. To our knowledge, this is the first time in literature that implantation of allogeneic Ad-MSCs around lacrimal glands has been found as an effective therapeutic alternative to treat dogs with KCS. These results could reinforce a good effective solution to be extrapolated to future studies in human.

Figures

Figure 1

Cell morphology and proliferative curve of canine Ad-MSCs cultures. (a) In primary cultures, a large number of adherent cells with fibroblastic morphology were observed from the first day of culture, forming frequent CFUs. On secondary culture, canine Ad-MSCs appeared as spindle-shaped cells that were grown in a monolayer. Bars 200 μm. (b) Representative curve obtained with MTS cell proliferation assay at p2 showing from 24 hours an important proliferative capacity, initiating the logarithmic growth phase, and reaching its plateau phase around 14 days.

Figure 2

Representative immunophenotype profiles of canine Ad-MSCs for several mesenchymal and hematopoietic markers. FACS analysis revealed a homogeneous cell population, characterized by the positive expression of CD29, CD90 and STRO-1, and lack expression of CD34, CD45 and major histocompatibility class II (MHC-II).

Figure 3

Assessment of trilineage differentiation. (a) Control. Cells maintained in control medium for 21 days. (b-c) Assessment of osteogenic differentiation. (b) Histochemical localization of ALP. Osteoinduced cells formed numerous nodules highly positive for ALP staining. (c) Positive Alizarin Red S staining by day 21 whereas red calcium nodules clearly appeared on the osteoinduced cultures. (d) Assessment of adipogenic differentiation. Positive Oil Red O staining by day 21 confirmed the presence of lipid droplets only in adipogenic-induced cells. (e-f) Assessment of chondrogenic differentiation. Histological sections of pellets after 21 days in the presence or absence of rhTGF-β1. (e) Control pellets incubated without rhTGF-β1. (f) Pellets incubated with rhTGF-β1 clearly displayed improved chondrogenesis with positive toluidine blue staining. Bars 200 μm in (a–d) and 100 μm in (e-f).

Figure 4

Photographs of the right eye of dog number 7 with chronic KCS and STT 0 mm/min during 2 years, severe ocular discharge, severe hyperaemia, and moderate cornea changes (edema, cornea opacity, and vascularization). (a) Eye at baseline (0). (b) Same eye at 9 months after cell implantation. STT 19 mm/min, without secretion, mild hyperaemia, and improvement on cornea changes and showing a good lacrimal meniscus.

Figure 5

Mean values ± standard deviations of the ocular parameters scored during the 9-month follow-up after treatment. For Schirmer test, tears production was measured at different time points (a). For (b), (c), and (d) mean scores are represented. Stars indicate statistically significant differences in comparison with baseline. P < 0.01 (∗∗), and P < 0.001 (∗∗∗). There were no differences among the other groups in any case.