We generated immortalized BMSC clones

We generated immortalized BMSC clones to perform detailed analysis of functional diversity in BMSC subtypes to levels not previously described. Similar immortalization techniques (e.g., hTERT combined with human papillomavirus E6/E7) have been used for the clonal analysis of BMSCs, which found OAC, AO, OC, unipotent, and nullipotent clones at varied frequencies, but other functional characteristics and discriminating features were not examined (Larsen et al., 2010; Okamoto et al., 2002). Notably, however, enhanced immune-related features could be assigned to non-bone-forming hTERT-BMSCs in a previous study (Larsen et al., 2010). Using non-immortalized BMSC clones, Muraglia et al. identified OAC-, OC-, and O-competent lines only, with very rare (<1%) nullipotent subsets (Muraglia et al., 2000). Rather than determine occurrence of O/A/C phenotypes in heterogeneous BMSCs, here our aim was to identify molecular and biophysical markers of BMSC subtypes and their alignment with non-progenitor cell functions, including immunomodulatory properties. To this end, from the original hTERT-BMSC lines generated, we selected four clones positioned at opposing ends of a differentiation competency scale, tripotent Y101/Y201 and relatively impotent Y102/Y202, for in-depth cell profiling. The OAC-differentiating lines expressed an extensive and significantly enriched gene set consistent with blood vessel support/interaction compared to nullipotent clones. This may reflect a relationship between BMSC differentiation competency and a perivascular niche location (Sacchetti et al., 2007). Conversely, Y102 and Y202 lines expressed CD online widely used to describe BMSCs (Dominici et al., 2006; Pittenger et al., 1999) but exhibited very little differentiation capacity. Subsequent assays revealed that an equivalent CD317+ cell type existed in heterogeneous primary BMSC populations at ∼1%–3% frequency and CD45−CD31−CD317+ cells represented ∼0.4% of whole human BM-MNCs. Primary cell analysis and in vivo validation is an important component of our study. Data obtained using in vitro immortalized cell lines must be interpreted with some caution, considering the effects of hTERT transduction artifacts. Integration site, enhanced telomerase activity and long-term culture are all likely to impact on cell function. We attempted to minimize potential confounding effects by selecting two cell lines, each displaying similar properties (Y101/Y201 and Y102/Y202). We used Raman spectroscopy, which has been demonstrated as a non-destructive label-free method to study the biological state of single cells and to identify molecular-scale differences for cell discrimination (see Schulze et al., 2010, for example). In this work, Raman spectroscopy was able to uniquely identify the four hTERT-BMSC clones, separating Y101 from Y201 and confirming a striking biomolecular relationship between Y102/Y202 and primary CD317+ cells, which also shared an enlarged, spread morphology, forming compact, non-migratory colonies. We performed detailed bioinformatics to scrutinize gene expression profiles against the parental BMSCs. These analyses ultimately identified the closely clustered Y102 and Y202 lines as having remarkably high basal level expression of factors associated with a pro-inflammatory and anti-viral function. The significantly enriched gene sets we identified in unchallenged Y102/Y202 BMSC lines are typical of an immediate immunostimulatory response to infection and other insults. Notably, all BMSC clones exhibited similar inhibitory effects on PBMC proliferation when activated by exposure to anti-CD3 and anti-CD28 to mimic antigen presentation. Consequently, Y102/Y202 BMSC subtypes may function in both immunostimulatory (early, pro-inflammatory) and immunosuppressive (anti-inflammatory, tissue protective) responses depending on the immune environment; however, functional validation and further refinement of the characteristics of this CD317-expressing population is required. Dual immunomodulatory functions for BMSCs have been identified and discussed (Dazzi and Krampera, 2011), but not assigned to BMSC subtypes. CD317+ cells (as well as Y102/Y202) were characterized as having elevated IL-7 expression, which has well-established roles in T and B cell development (Ceredig and Rolink, 2012). Examination of skeletal tissues in a lineage-tracing mouse model confirmed that a substantial subpopulation of the differentiated progeny of BMSCs could not have arisen from IL-7-expressing stromal progenitors. It should be noted that differentiated skeletal cells may express IL-7 (Long et al., 2008; Zhu et al., 2007), which would also be revealed as EYFP positivity. However, these studies provide unequivocal evidence linking IL-7 expression in stromal subsets with differentiation incompetence during normal development in vivo. Our in vivo observations were reflected in our in vitro assays of differentiation, where the IL-7hi immortalized BMSC clones (Y102/Y202) exhibited limited skeletogenic potential. These in vitro assays are prone to artifact that could result in non-specific detection of widely used markers of differentiation that may not report authentic in vivo differentiation capacity, observed for example using in vivo transplantation techniques (Robey et al., 2014). Even with these caveats, we still failed to detect clear evidence of differentiation ability in Y102/Y202 CD317+ IL-7hi BMSC lines. In vivo, CD317+ cells had a dispersed bone marrow distribution and did not generally colocalize with Cxcl12-DsRed+ perivascular stromal cells, again pointing to an alternative function for CD317+ BMSCs, unrelated to CXCL12-mediated hematopoietic stem cell niche support. CD317 has a well-defined anti-viral function acting to tether and restrict the release of viral particles and may also trigger the viral immune response (Hotter et al., 2013). The existence of a marrow-resident, non-migratory, non-differentiating colony-forming stromal cell type, apparently primed for host defense, is an interesting concept. These cells could act as first responders to pathogen invasion and/or provide low-level immune control. As IL-7 has been implicated as a causative factor in autoimmunity, including rheumatoid arthritis (Dooms, 2013), unregulated control of CD317+ cell numbers and function may also contribute to disease onset and progression. These findings highlight the importance of identifying unique markers of BMSC sub-populations and assigning function, which will instruct strategies to analyze stromal-immune cell interaction, differentiation competency, and cell selection for safe and effective therapy.