Tissues from equine cadaver ligaments up to 72 hours of post-mortem: a promising reservoir of stem cells

Abstract

BackgroundMesenchymal stem cells (MSCs) harvested from cadaveric tissues represent a promising approach for regenerative medicine. To date, no study has investigated whether viable MSCs could survive in cadaveric tissues from tendon or ligament up to 72 hours of post-mortem. The purpose of the present work was to find out if viable MSCs could survive in cadaveric tissues from adult equine ligaments up to 72 hours of post-mortem, and to assess their ability (i) to remain in an undifferentiated state and (ii) to divide and proliferate in the absence of any specific stimulus.MethodsMSCs were isolated from equine cadaver (EC) suspensory ligaments within 48–72 hours of post-mortem. They were evaluated for viability, proliferation, capacity for tri-lineage differentiation, expression of cell surface markers (CD90, CD105, CD73, CD45), pluripotent transcription factor (OCT-4), stage-specific embryonic antigen-1 (SSEA-1), neuron-specific class III beta-tubulin (TUJ-1), and glial fibrillary acidic protein (GFAP). As well, they were characterized by transmission electron microscope (TEM).ResultsEC-MSCs were successfully isolated and maintained for 20 passages with high cell viability and proliferation. Phase contrast microscopy revealed that cells with fibroblast-like appearance were predominant in the culture. Differentiation assays proved that EC-MSCs are able to differentiate towards mesodermal lineages (osteogenic, adipogenic, chondrogenic). Flow cytometry analysis demonstrated that EC-MSCs expressed CD90, CD105, and CD73, while being negative for the leukocyte common antigen CD45. Immunofluorescence analysis showed a high percentage of positive cells for OCT-4 and SSEA-1. Surprisingly, in absence of any stimuli, some adherent cells closely resembling neuronal and glial morphology were also observed. Interestingly, our results revealed that approximately 15 % of the cell populations were TUJ-1 positive, whereas GFAP expression was detected in only a few cells. Furthermore, TEM analysis confirmed the stemness of EC-MSCs and identified some cells with a typical neuronal morphology.ConclusionsOur findings raise the prospect that the tissues harvested from equine ligaments up to 72 hours of post-mortem represent an available reservoir of specific stem cells. EC-MSCs could be a promising alternative source for tissue engineering and stem cell therapy in equine medicine.

Ligament-Derived Stem Cells: Identification, Characterisation, and Therapeutic Application

“…Osteogenic, adipogenic, and chondrogenic differentiation has also been demonstrated in other ligament types, including LDSCs isolated from equine suspensory [16], human interspinous [19], human medial collateral [20], and human posterior cruciate [21] ligaments, as well as LDSCs derived from canine CCL in our hands (Figure 2). …”

“…However, there are a number of other studies which have isolated and cultured LDSCs from other species, including horses [16], pigs [17], and rabbits [18], as well as other ligament types, including rabbit medial collateral ligament (MCL) [18] and human interspinous ligament [19]. …”

“…Despite the different approaches to LDSC isolation, cells obtained through tissue digestion or tissue explants seem to demonstrate similar stem cell characteristics [22]. Cells are then cultured in vitro and can be extensively expanded up to 25 population doublings [21] or 20 passages [16]. Unlike other stem cell types, there is little research on the optimum culture conditions for LDSC survival and expansion.…”

“…LDSCs isolated from ACL and posterior cruciate ligament (PCL) demonstrated a fibroblastic morphology [15, 21] and those from MCL a polygonal morphology [18, 20]. LDSCs isolated from equine suspensory ligaments (SLs) demonstrated a heterogeneous morphology, with predominantly fibroblastic cells, but also with the presence of smaller rounded cells [16]. We have observed both rounded and fibroblastic cell morphologies upon initial seeding of stem cells derived from canine cranial cruciate ligament (CCL); however, the rounded morphology was lost with passaging (Figure 1).…”

Monoclonal Antibodies Specific to Water Buffalo (Bubalus bubalis) Myxovirus Resistance Protein_1

“…Pieces of tissue (~50g) were recovered using scalpel and forceps, dipped in 10% iodine polyvidone solution, and washed three times with phosphate-buffered saline (PBS, Lonza) supplemented with 1% penicillin-streptomycin (Gibco) and 1% Amphotericin B (Gibco). Subsequently, tissue samples were cut into small fragments (2-5 mm 3 ) under aseptic conditions, washed twice with PBS, and processed as described [22]. Briefly, fragments were digested for 18h at 37°C and 5% CO2 in a solution containing 0.2 % (w/v) collagenase A (Roche) in PBS supplemented with 1 % penicillinstreptomycin and 1% Amphotericin B.…”