Update on canine MSC markers
2014-03-27

Degeneration of the intervertebral disk (IVD) is a major cause of low back pain. Low back pain is one of leading causes of work disability and hence a public health concern that warrants improved treatment strategies 1. Recently, cell-based regenerative therapies have emerged for treatment of the degenerated IVD. In this respect, dog develops spontaneous IVD degeneration and disease and hence is a valuable preclinical large animal model. Bone marrow- and adipose tissue-derived mesenchymal stem cells (MSCs) have already been applied for disk repair in experimental dogs and even in human phase I and II clinical trials. Here, we summarize existing and present novel antibodies and introduce the cognate antigens as biomarkers for the characterization of canine MSCs. Surgical therapies of IVD disease are salvage procedures that are generally successful but are associated with various complications, such as spinal instability, adjacent segment degeneration, and failure of surgical implants 2, 3. This has prompted interest in (mesenchymal stem) cell-based regenerative strategies aiming at restoring the health and function of the IVD, which can be applied at an earlier stage of IVD degeneration. MSC-based therapies are being developed and further evaluated for their safety and efficacy mainly in animal models, in which IVD degeneration is experimentally induced. Ideally, in a preclinical phase regenerative strategies should be performed in animal models that spontaneously develop IVD degeneration and disease. Dogs can suffer from back pain and neurological deficits as a result of IVD disease, which results in a strikingly similar clinical representation as observed in humans. Moreover, dogs show similar biomechanical properties and biomolecular and histological changes as humans during IVD degeneration 4. Bone marrow- and adipose tissue-derived MSCs have already been applied for IVD repair in experimental dogs 5, 6. In humans with low back pain, phase I-II clinical trials have been initiated using MSCs for IVD repair (http://clinicaltrials.gov/show/NCT01290367). Despite of considerable progress, the cellular and molecular basis of these successful interventions are only poorly understood. In particular, it is still an open question, which MSC subsets are mainly responsible for IVD regeneration. In humans, several subsets of bone marrow-derived MSCs with differential capacities to give rise to chondrocytes and adipocytes were identified using a large panel of antibodies against cell surface antigens 8-10. In this work, we present a summary of antibodies originally raised against human MSC antigens that cross-react with their canine counterparts. These tools may facilitate the comparison of human and canine MSCs in vitro and in vivo and, therefore, accelerate the research in the field of IVD regeneration. In a recent report, Rozemuller et al. 11 described the prospective isolation of MSCs from multiple mammalian species using cross-reacting anti-human monoclonal antibodies 11. They identified seven antibodies which cross-reacted with canine targets including a commercially available antibody against CD271 and the in-house generated antibodies W8B2 (MSCA-1, TNAP), W4A5 (orphan), 39D5 (CD56), W3C4 (frizzled-9, CD349), W5C4 (orphan), and 58B1 (once orphan, now CD276). Here, we show the reactivity profiles of additional selected antibodies on canine bone marrow- (Fig. 1A) and adipose tissue-derived MSCs (Fig. 1B), which were analyzed after passage 1 of culture. As shown in Figures 1A and 1B, the cross-reactive binding of antibodies W8B2, 39D5, W3C4, W5C4, and 58B1 could be confirmed in both tissues. In addition, flow cytometry analysis revealed that the CD34-reactive antibody 43A1 recognizes the canine counterpart as well (Fig. 1B). In line with the fact that CD34 is expressed on adipose tissue-derived MSCs but not on bone marrow-derived MSCs 12, antibody 43A1 recognizes canine MSCs form adipose tissue but not from bone marrow. The specificity of the commercially available antibody FM276 and our antibodies 58B1, HEK5-1B3, T-1A5, and IPS-K-2B6A8 for CD276 was confirmed by their selective reactivity with NIH-3T3 cells transfected with human CD276 (Fig. 1C) and their negative reactivity with parent NIH-3T3 cells. Figure 1C additionally shows that all antibodies recognize both the long and the short isoform of CD276. Of these, only clone FM276 does not cross-react with canine CD276 (Figs. 1A and 1B). The immunostaining profiles of all tested antibodies of bone marrow- as well as of adipose-derived MSC is additionally shown in Supporting Information Figure. The described antibodies against canine MSC antigens summarized in Table 1 may present valuable tools to identify biomarkers for the profiling of MSC populations and identify the most potent subpopulation of MSCs to be used for the successful treatment of IVD degeneration in the dog as an experimental and clinically relevant animal model. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.