Acta Histochemica 119 (2017) 150–160 Contents lists available at ScienceDirect Acta Histochemica jo ur nal homepage: www.elsevier.de/acthis RNA in situ hybridization characterization of non-enzymatic derived bovine intervertebral disc cell lineages suggests progenitor cell potential Petra Kraus, Rachel Yerden, Victoria Kocsis, Thomas Lufkin ∗ Department of Biology, Clarkson University, Potsdam, NY 13699, USA a r t i c l e i n f o Article history: Received 21 October 2016 Received in revised form 19 December 2016 Accepted 21 December 2016 Keywords: RNA in situ hybridization Regenerative medicine Single cell quality control Annulus fibrosus Nucleus pulposus a b s t r a c t Degeneration of the intervertebral disc (IVD) is a meritorious target for therapeutic cell based regenerative medicine approaches, however, controversy over what defines the precise identity of mature IVD cells and lack of single cell based quality control measures is of concern. Bos taurus and human IVDs are histologically more similar than is Mus musculus. The mature bovine IVD is well suited as model system for technology development to be translated into therapeutic cell based regenerative medicine applications. We present a reproducible non-enzymatic protocol to isolate cell progenitor populations of three distinct areas of the mature bovine IVD. Bovine specific RNA probes were validated in situ and employed to assess fate changes, heterogeneity, stem cell characteristics and differentiation potential of the cultures. Quality control measures with single cell resolution like RNA in situ hybridization to assess culture heterogeneity (PISH) followed by optimization of culture conditions could be translated to human IVD cell culture to increase the safety of cell based regenerative medicine. © 2016 Elsevier GmbH. All rights reserved. 1. Introduction With an increase in average life expectancy, modern society faces the challenges and burdens of age related medical conditions like intervertebral disc (IVD) degeneration. Regenerative medicine (RM) seeks to address these problems via cell-based approaches, replacing ailing cells, tissues or entire organs. Autologous mes- enchymal stem cells (MSC) are of increasing interest to the field (Risbud et al., 2004b; Sakai and Andersson, 2015) circumvent- ing ethical issues associated with embryonic stem (ES) cells and less likely associated with tumorigenicity than induced pluripo- tent stem (iPS) cells (Takahashi and Yamanaka, 2006). Several in vivo niches for MSCs have been identified over the years, with bone marrow MSCs likely being one of the most studied ones (Bara et al., 2014). Like for any organ targeted for RM, the objective to direct patient-specific cells towards an IVD fate is appealing, yet for safety reasons, there is a desperate need for single cell resolution quality control measures, especially in the wake of reported malpractice associated with an increase in stem cell clinics and a flourishing ∗ Corresponding author at: Department of Biology, Clarkson University, 8 Clarkson Avenue, Potsdam, NY 13699-5805, USA. E-mail address: tlufkin@clarkson.edu (T. Lufkin). stem cell tourism market (Berkowitz et al., 2016). Identification of characteristic IVD lineage markers is important, yet none has been conclusively identified to date (Lv et al., 2014). Commonly RT- PCR or microarray expression profiling on pooled cells is applied to identify IVD cells in situ or in vitro (Arufe et al., 2010; Lee et al., 2015; Liu et al., 2011; Lv et al., 2014; Minogue et al., 2010; Thorpe et al., 2016). This pooling could mask heterogeneous cell populations and cause misidentification of cells. The IVD is suited for RM approaches, as ailing discs might be identified long before an acute medical problem arises, allow- ing to generate autologous cells in culture. IVDs are the largest non-vascularized structures in our body (Moore, 2006), acting as semi-movable joints between the vertebral bodies (VB) providing flexibility and load transmission in the spine (Michalek and Iatridis, 2012; Moore, 2006). The mature IVD consists of minimally four dis- tinct tissue types: 1) The shock absorbing nucleus pulposus (NP); 2) the ringed exteriorly positioned annulus fibrosus (AF); 3) the transition zone (TZ) which is positioned between the NP and the AF and; 4) the endplates which sandwich the NP, TZ and AF to adja- cent VBs (Pooni et al., 1986). Anatomically, mature IVDs appear as simple organs, but patterning events during embryogenesis result- ing in different mesoderm derived cell lineages and structures are complex (Bara et al., 2014; Choi et al., 2008; Choi and Harfe, 2011; Choi et al., 2012; Maier and Harfe, 2011; Maier et al., 2013; Risbud et al., 2010; Sivakamasundari and Lufkin, 2012; Smith et al., 2011) http://dx.doi.org/10.1016/j.acthis.2016.12.004 0065-1281/© 2016 Elsevier GmbH. All rights reserved.