Mesenchymal Stem Cells - Appropriate Use in Equine Joint Disease

Take Home Message: Mesenchymal stem cells (MSCs) provide the potential to regenerate tissues without the production of scar tissue that is generally associated with healing in musculoskeletal tissues. At the moment , bone marrow-derived MSCs have received the most validation including de monstration of effectiveness intraarticularly to enhance soft tissue healing as well as improving articular cartilage repair. There is some evidence for usefulness with osteoarthritis (OA) and efficacy a lso demonstrated in augmentation of healing of injuries to the superficial digital flexor tendon as well as the suspensory ligament.

I. INTRODUCTION

Stem cells continue to receive much scientific attention as well as coverage in the lay press. The principal reason is that they provide the potential to regenerate tissues without the production of scar tissue that is generally associated with healing in musculoskeletal tissues. The goal in the therapeutic use of MSCs in musculoskeletal disease is to harness the regenerative nature of these cells focusing on the potential to grow new tissues and organs to replace damaged or diseased tissue.¹

The term was first coined as a synonym for a mitotically quiescent primordial germ cell.² Stem cells have also been described as the natural units of embryonic generation or adult regeneration of a variety of tissues.³ In the1960s bone marrow-derived cells were identified as being capable of differentiating into cells (osteoblasts) of mesenchymal origin.⁴ The recognition that these bone marrow-derived MSCs (BMSCs) can differentiate into cells of different lineages has now been widely established^5-8 and more recently this has been recognized with equine BMSCs.^9,10 Pittenger et al described MSCs as “multipotent cells that are present in adult bone marrow that can replicate as undifferentiated cells and have potential to differentiate to lineages of mesenchymal tissue including bone, cartilage, muscle, ligament, tendon, adiposeandstroma”.⁶ The multiple different pathways of multipotent MSCs and the proteins involved in their transcriptional control have been described in a review of MSC therapy in equine musculoskeletal disease.¹ For instance, Runx2 is the principal protein that mediates the transcriptional control of osteogenesis, Sox9 is the equivalent for chondrogenesis and Pparγ is the equivalent for adipogenesis (not that we generally want to grow fat!). MyoD is a regulatory factor that controls myogenesis. Smad8 and scleraxis are transcriptional factors thought to be important in tenogenesis.

II. TYPES OF STEM CELLS

Embryonic stem cells: These are derived from the inner cell mass of any early developing embryo and have plasticity and potentially unlimited capacity for self-renewal.¹¹ A company in the United States has been working on fetal-derived stem cells and has shown encouraging results with collagenase-induced superficial digital flexor (SDF) tendonitis.¹²

Induced pluripotential cells: Transfection of somatic cells with four separate genes (Oct-3/4, Sox2, Klf4, cMyc) potentially provides cells capable of regenerating every cell in the body.¹³ Investigation with equine induced pluripotential cells is being done but is not a clinical tool at the moment.

Adult-derived stem cells: As mentioned before, these are small populations of cells capable of differentiating into different cell lines under the right conditions. They commonly occupy a perivascular niche¹⁴ and may indeed be pericytes.¹⁵ In the musculoskeletal field, such cells have been identified in the surface layer of articular cartilage, synovial membrane, bone marrow, tendon, skeletal muscle, adipose tissue and umbilical cord. In addition to providing hemopoietic cells, stem cells responsible for replenishing the cells of the blood, bone marrow contains a different group of cells that form the fibrous “stroma” within the marrow and provide a source of cells, the mesenchymal stromal cell or mesenchymal progenitor cell, which acts as “general repair man” for injuries to any of the mesenchymal tissues within the body, gaining access via the systematic blood supply.¹¹ They are believed to be attracted via chemokines or different cell surface receptors expressed after injury. It has been noted that a small number of cells could still influence repair significantly and their “paracrine” effects are increasingly influencing repair rather than their ability to differentiate into the target cell and synthesize new tissue themselves.

There is recent evidence that, in addition to providing cells, MSCs exert trophic and immunomodulatory activities that enhance the local regenerative microenvironment.¹⁴ It has been suggested that MSCs from mature animals decline with age and their ability for successful and functional differentiation may also decline with age.¹⁶ However, it has been recently demonstrated that adult equine MSCs produce cartilage-like extracellular matrix (ECM) at the same level as MSCs derived from two- to four-month old foals whereas that both groups of BMSCs produce superior cartilage-like neo-tissue than either young or adult chondrocytes.¹⁷ The clinical use of MSCs in horses, justification for their use and issues surrounding their use has been reviewed.¹⁶

III. RECOVERY TECHNIQUES FOR MESENCHYMAL STEM CELLS

Isolation of MSCs from the marrow or digestive tissue extracts is most commonly achieved by simple adhesion and proliferation of MSCs to culture surfaces. This achieves a significant, if not homogenous, MSC population. Near-homogenous MSC populations have been reported from adhesion sorting.⁶ Research continues on more rigorous methods of identifying stem cells through use of cell surface antigens such as cluster differentiation (CD) factors 34 and 44. The temporal gene and protein expression changes during establishment of equine MSC cultures have been described.¹⁸ This study demonstrated numerous dynamic changes in cell surface molecule expression during early establishment of MC culture populations, which may help improve mesenchymal precursor cell (MPC) isolation techniques for research or therapeutic applications.

IV. HISTORY OF STEM CELLS IN EQUINE ORTHOPAEDICS

The first use was bone marrow aspirates (these contain a low amount of MSCs – approximately 2x103/ml). This was followed by the use of adipose stromal vascular fraction (SVF) with the original application being Vet-Stem^a in the United States. This generates 5–10x106 nucleated cells but only 2–4% will be MSCs. A similar product is now available in Australasia. Bone-derived culture-expanded MSCs have been used in several laboratories and based on research at Colorado State University (CSU), The Royal Veterinary College and Cornell University and two commercial bone-derived culture-expanded MSC units grown from this: VetCell Bioscience Ltd.^b in the United Kingdom and Advanced Regenerative Therapies (ART)^c in Colorado. Because bone marrow contains 1,000– 2,000 MSCs/ml, culture expansion is necessary to obtain clinically relevant numbers. The culture procedure involves the seeding of nucleated cells into tissue culture flasks, allowing the colonies of stem cells to form in six to nine days and culture to expand to obtain approximately 10 million cells in two weeks and 20 million cells in three weeks. At CSU, when the cells are going to be used immediately, they are suspended in phosphate- buffered saline for injection (HA is often given concomitantly when MSCs are administered to joints). If stem cells are being transported off campus they are preserved in autologous serum and dimethyl sulphoxide and transported on dry ice. Treatments are typically administered for conditions that have not responded to established treatment modalities.

V. BONE MARROW VERSUS ADIPOSE TISSUE AS A SOURCE OF MSCS

Most research for clinical treatment in human and veterinary orthopaedics has used autologous MSCs mainly from bone marrow.¹⁹ Adipose tissue is readily available and a normal amount of research has been done on fat as a source of MSCs.²⁰ The current evidence, at least in the horse, is that while adipose-derived MSCs can differentiate into musculoskeletal tissues, they are inferior to bone marrow under current differentiation conditions. Equine-specific research supports this concept.^21-23 In comparisons using the same cell culture conditions, BMSCs have been shown to be more chondrogenic with regard to extracellular matrix production of both collagen and proteoglycans (glycosaminoglycans).²¹ With appropriate manipulation, adipose-derived cells can begin to differentiate into musculoskeletal tissue in similar (but not superior) fashion with the addition of BMP-6.²⁴

VI. CLINICAL USE OF ADIPOSE SVF

The first commercial product in the United States was based on adipose-derived SVF by Vet-Stem. There are two publications in dogs (a more recent target population) showing promising results: one study was in elbow osteoarthritis (OA) and the other was in coxofemoral joint arthritis (the latter study was a randomized, double blinded study)^25,26 but no equine clinical results have been reported. However, positive results have been shown with collagenase-induced SDF tendonitis in a short-term pilot study.²⁷ Adipose SVF evaluated in collagenase-induced SDF tendonitis improved histologic scores compared with controls.

VII. USE OF BMSCS FOR TREATMENT OF JOINT-RELATED DISEASES IN HORSES

Early work using labelled MSCs has shown that they have an affinity for damaged joint tissue and more recent work has confirmed their ability to localize and participate in repair of damaged joint structures including cruciate ligaments, menisci and articular cartilage lesions.²⁸ Most in vivo studies done in animals other than horses have focused on meniscal repair. [...]