A reproducible method for the isolation and expansion of ovine mesenchymal stromal cells from bone marrow for use in regenerative medicine preclinical studies

Abstract

The use of multipotent mesenchymal stromal cells (MSCs) as candidate medicines for treating a variety of pathologies is based on their qualities as either progenitors for the regeneration of damaged tissue or producers of a number of molecules with pharmacological properties. Preclinical product development programmes include the use of well characterized cell populations for proof of efficacy and safety studies before testing in humans. In the field of orthopaedics, an increasing number of translational studies use sheep as an in vivo test system because of the similarities with humans in size and musculoskeletal architecture. However, robust and reproducible methods for the isolation, expansion, manipulation and characterization of ovine MSCs have not yet been standardised. The present study describes a method for isolation and expansion of fibroblastic-like, adherent ovine MSCs that express CD44, CD90, CD140a, CD105 and CD166, and display trilineage differentiation potential. The 3-week bioprocess proposed here typically yielded cell densities of 1.4 × 10 MSCs/cm at passage 2, with an expansion factor of 37.8 and approximately eight cumulative population doublings. The osteogenic potential of MSCs derived following this methodology was further evaluated in vivo in a translational model of osteonecrosis of the femoral head, in which the persistence of grafted cells in the host tissue and their lineage commitment into osteoblasts and osteocytes was demonstrated by tracking enhanced green fluorescent protein-labelled cells. Copyright © 2016 John Wiley & Sons, Ltd.

Clinical translation of a mesenchymal stromal cell-based therapy developed in a large animal model and two case studies of the treatment of atrophic pseudoarthrosis

“…With respect to the scaffold, commercial particulated deantigenized bone matrix from human cadaveric donors was used as a natural osteoinductive and osteoconductive agent. This approach was preferred provided that cells attached to the scaffold would persist longer in the site of administration, as was demonstrated in a sheep model of avascular osteonecrosis (Caminal et al ., ). Animals included in the preclinical study weighted 37.1 ± 3.5 kg (range 30.4–41.8 kg, n = 16).…”

“…Both ovine and human tissues were harvested, fixed in 4% formalin at room temperature, decalcified in 5% formic acid (Decalcifier I; Surgipath Canada Inc., Winnipeg, Canada), and embedded in paraffin for subsequent histological and immunohistochemical procedures (Caminal et al ., ; Oliver‐Vila et al ., ). Microtome sections 4 μm thick were either stained with H&E or immunostained for the detection of macrophages (CD68, KP‐1; Dako, Glostrup, Denmark), and vessels (endothelial cells, CD31, JC70A, Dako), as reported elsewhere (Gomez‐Puerta et al ., ).…”

Mammalian MSC from selected species: Features and applications

“…Bone marrow waste material is available from elective knee and hip surgery or major amputations and moreover could be gained from voluntary crista iliaca punctures. Isolation procedures are similar as compared to methods from other species like dog , horse , or sheep and aim at enrichment of MSC from the mononuclear cell fraction. In rodents, the isolation procedure is modified, taking into account the low amount of bone marrow available at all.…”

Osteogenic commitment of Wharton’s jelly mesenchymal stromal cells: mechanisms and implications for bioprocess development and clinical application

“…Although systemic infusion of MSC has been shown to increase bone growth and repair in clinical trials [53][54][55], administered cells engraft poorly. In this line, previous in vivo studies carried out by our group in which ovine eGFP+ BM-MSC were infused in an ovine model of osteonecrosis of the femoral head demonstrated the presence of non-stained eGFP osteocytes in newly formed bone matrix, suggesting that contribution of MSC lies also in paracrine signalling that activate and recruit host osteoblasts to the bone repair areas [56]. Increasing evidence have shown that nanosized, membrane-encapsulated EVs are one of the most active MSCs' secreted factors [25].…”