Mesenchymal Stromal Cells (MSC) are a subset of heterogeneous fibroblastoid stromal cells with high self-renewal capacity and differentiation potential. Over the last years, the interest on MSC in human and veterinary medicine has dramatically risen. This is confirmed by the abundance of clinical trials aimed at validating their efficacy in a wide range of pathological conditions (https://clinicaltrials.gov/) and by the increasing number of reports concerning MSC clinical use in spontaneous animal diseases (Voga et al., 2020). By virtue of their biological features, MSC are currently the most extensively investigated cell type for advanced therapies. The ability to modulate inflammation/immunologic related disorders, the pro-regenerative potential, the tropism for injured sites, and the paracrine signaling make them suitable "smart" therapeutic tools (Pittenger et al., 2019).
Over time, MSC have been collected from many different tissues and organs of adult mammals, like bone marrow, peripheral blood, synovial fluid, umbilical cord blood, Wharton's jelly, placenta, spleen, adipose tissue (Han et al., 2019).
In a position paper of the International Society for Cellular Therapy – ISCT, the main basic features of MSC were elucidated (Dominici et al., 2006). According to ISCT suggestions, MSC must display the ability to adhere to plastic when isolated from tissues and cultured in vitro, must express several antigens such as CD90, CD73, CD105 in a percentage ≥ 95%, and must express the typical hematopoietic surface molecules (CD45, CD34, CD14) in a rate ≤ 5%. Finally, they must be multipotent in vitro and display adipocytic, osteoblastic and chondroblastic trilineage differentiation potential (Dominici et al., 2006).
MSC are becoming increasingly used in canine species to treat a range of diseases, including orthopedic (Dias et al., 2021), digestive tract disorders (Cristóbal et al., 2021; Pérez-Merino et al., 2015), as well as diseases of the liver (Gardin et al., 2018), kidney (Lee et al., 2017), heart (Pogue et al., 2013), respiratory, skin (Enciso et al., 2019), ocular (Villatoro et al., 2015) and reproductive system. In addition, canine MSC have been extensively studied due to the interest this species holds as preclinical/clinical models for cell therapy in humans (Hoffman & Dow, 2016).
Different research groups have carried out canine MSC isolation. Their characterization is usually performed through flow-cytometry, immuno-fluorescence, or RT-PCR. Comparison between various studies revealed some differences concerning ISCT indications for surface markers of MSC. The most recurrent positive markers for canine MSC are CD29, CD44 and CD90 (Ivanovska et al., 2017; W. S. Lee et al., 2011; Russell et al., 2016; Takemitsu et al., 2012); among these, only CD90 is included in ISCT guidelines. CD73 was detected by flow cytometry in canine MSC only by Russel et al. (Russell et al., 2016), and through RT-PCR in three published works (Ivanovska et al., 2017; W. S. Lee et al., 2011; Screven et al., 2014). Canine MSC expression of CD105 marker was detected by flow cytometry only in two papers (Bearden et al., 2017; Chow et al., 2017).
Different sources of MSC are currently used in dogs, but adipose tissue (AT) is one of the most studied since it is abundant, easy to collect using minimally invasive procedures, and rich in MSC that are relatively easy to isolate, propagate and exhibit high proliferative potential in vitro (Rashid et al., 2021; Zhan et al., 2019).
Isolation of MSC from AT is commonly carried out by a standardized procedure including the following phases: 1. Mincing tissue with scissors or scalpel; 2. Digestion by collagenase type I for 45 min to 1 h at 37 ◦C by gently shaking in a water bath; 3. Centrifugation and removal of the floating lipid layer; 4. Filtration of the stromal vascular fraction (SVF) through 100 and 70, or 40 µm filters; 5. Washing and new centrifugation; 6. Removal of the supernatant, resuspension of the cell pellet, and seeding in a culture flask. 7. Removal of non-adherent cells from the culture 48 hours after seeding. Comparing different protocols described in the relevant literature, it is evident that the main differences concern collagenase concentration and digestion length, but no other consistent differences emerge. The procedure described in the most of the studies, therefore, represents a succession of consolidated phases which is repeated rather constantly in different laboratories (Bearden et al., 2017; Chow et al., 2017; Ivanovska et al., 2017; W. S. Lee et al., 2011; Russell et al., 2016; Screven et al., 2014; Takemitsu et al., 2012; Zhan et al., 2019).
In our multi-year experience in isolating and expanding AT-derived MSC from animal species, we observed that vascular-stromal fragments, usually eliminated by filtration or discarded after 48h from seeding, release over time cells able to adhere to the plastic and to proliferate for subsequent passages. Two possible hypotheses have been formulated to explain this behavior: i) Cells adhere at different time points but share the same MSC biological identity; ii) Cells adhere at different time points because they belong to different tissue niches. To verify the correct hypothesis, we compared MSC subpopulations from 3 donors to highlight differences or common traits. To this aim, 3 cell sub-populations were obtained based on their time-dependent affinity to plastic. They were compared for viability, proliferative activity, immune-phenotype, and differentiation ability towards adipogenic, osteogenic, and chondrogenic lineages.