Characterisation and differentiation potential of bone marrow derived canine mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) have potential for use in regenerative therapeutics, since they are capable of multi-lineage differentiation. In this study, primary canine MSCs (cMSCs) were isolated from bone marrow aspirates and characterised using marker expression and morphology. cMSCs expressed CD44 and STRO-1, but not CD34 or CD45.

Morphologically, cMSCs were similar to previously described MSCs and were capable of chondrocyte differentiation towards articular type cartilage, characterised by increased collagen type II vs. collagen type I expression and expression of Sox-9. cMSCs demonstrated no significant alterations in marker profiles and failed to differentiate into cardiomyocytes in response to a cardiac differentiation protocol or when co-cultured with canine cardiac stem cells. The study indicated that cMSCs can be derived readily from bone marrow and are capable of differentiation into articular cartilage, but appear to have limited ability to differentiate into cardiomyocytes using current protocols.

Introduction

Mesenchymal stem cells (MSCs) are adult stem cells that are lower in the stem cell hierarchy than embryonic stem cells. They exhibit a multipotent differentiation potential generally towards a limited number of cell types common to the tissue of origin, and maintain the capacity for self-renewal (Beltrami et al., 2007, Bajada et al., 2008).

Since there are no individual MSC specific markers, MSCs are defined based on three characteristics: (1) adherence to tissue culture plastic; (2) expression of certain surface antigen markers; and (3) ability to readily differentiate into different connective tissues (Pittenger et al., 1999, Dominici et al., 2006). A range of surface markers has been used to identify MSCs and generally includes expression of CD44 and STRO-1. MSCs mostly lack expression of the haematopoietic markers CD45 and CD34 and negative selection for these markers is used to exclude contamination with haematopoietic stem cells also present in the bone marrow compartment (Dominici et al., 2006).

CD44 is a glycoprotein surface marker expressed by MSCs isolated from different tissue sources and different species (Pittenger et al., 1999, Meirelles and Nardi, 2003, Kern et al., 2006). STRO-1 has been used to isolate a cell subset from human bone marrow with MSC properties (Simmons and Torok-Storb, 1991, Gronthos et al., 1994, Dennis et al., 2002). Alternative positive MSC markers include CD73, CD90 and CD105. Canine MSCs (cMSCs) have been reported as both positive and negative for CD90 and CD105, but negative for CD73, a classically positive MSC marker in humans; therefore marker expression in cMSCs shows some variability, suggesting that no one marker is correct (Kamishina et al., 2006, Seo et al., 2009, Oh et al., 2011). Furthermore, CD90 is expressed in canine haematopoietic stem cells (Lamerato-Kozicki et al., 2006).

Human MSCs can be readily induced to differentiate into osteoblast, adipocyte, and chondrocyte cell types in vitro (Gronthos et al., 1994, Yoo et al., 1998, Pittenger et al., 1999, Dennis et al., 2002, Suzdal’tseva et al., 2007). In comparison, few studies have utilised cMSCs, even though the dog is a good model for many human diseases and is frequently afflicted by bone and joint diseases. At the time of writing, cMSCs have been isolated from the bone marrow in only three studies in which the cMSCs were induced to differentiate toward osteogenic lineages by in vitro exposure to dexamethasone and co-culture with primary autologous osteoblasts, and towards chondrogenic lineage using scaffolds and co-culture; however, detailed characterisation of the cMSCs was not performed (Kadiyala et al., 1997, Csaki et al., 2007, Yang et al., 2011).

Demonstration of articular specific cartilage differentiation from cMSCs may facilitate the investigation of this cell source for the development of treatments for osteoarthritis (OA). Articular cartilage protects articular surfaces and participates in the fluid biomechanics of the joint. Damage to articular cartilage is a precursor to the development of OA (Cox and Cordell, 1977, Ghosh et al., 1990, Little et al., 1996). Chondrocytes isolated from stifle menisci and articular surfaces have been investigated as a direct source of differentiated cells for treatment of damaged cartilage in bovine and porcine models (Ibarra et al., 1997, Peretti et al., 2004). Despite some evidence of success, translation of this technique into clinical therapeutics has been limited by the difficulty in expanding chondrocytes in significant numbers due to the risk of de-differentiation towards fibro-chondrocytes during scale up in culture (Stokes et al., 2002). MSCs therefore offer a real alternative cell source for therapeutics in terms of chondrogenic potential (Wakitani et al., 1994, Wilke et al., 2007).

There is very little information available regarding the utility of cMSCs in therapy of other disease processes. In cardiovascular disease, it is known that the heart has a resident population of adult cardiac stem cells (CSCs), which may contribute to remodelling and repair; however, isolation of CSCs is technically challenging and generally yields low numbers of cells (Messina et al., 2004, Hodgkiss-Geere et al., 2012). Therefore, an alternative source of adult stem cells such as MSCs may yield greater numbers of cells from a mesodermal lineage similar to CSCs. Comparison of cell surface marker expression between MSCs and CSCs has been limited, since no stem cell markers common to these two lineages have been reported. Bone marrow derived stem cells have been induced to differentiate into cardiomyocyte-like cells in several species in vitro through treatment with the demethylating agent 5′-azacytidine (5′-AZA) (Makino et al., 1999, Shim et al., 2004, Xu et al., 2004, Kruglyakov et al., 2006, Antonitsis et al., 2007) (Appendix A; Supplementary Table S1). However, the ability of cMSCs to differentiate along the cardiac lineage in vitro has not been described previously.

The aim of this study was to isolate primary bone marrow derived cMSCs and to characterise them on the basis of morphological features, properties in culture and expression of surface markers. The chondrogenic and cardiogenic potential of the isolated MSCs was then evaluated by exposure to differentiation protocols and analysis of the resultant cell populations.