OECs and Schwann cells have been proposed as candidates for transplant mediated repair strategies of spinal cord injury. Previous studies have shown that they share a number of remarkably similar phenotypes, and growth factor responses [15, 22, 23, 27]. It is only recently that molecular studies have identified differences between these two cell types specifically in their interaction with astrocytes [17–19, 21, 28]. Furthermore, evidence suggests that other cell types, eg. fibroblast-like cells, previously rejected as contaminating cells, may have a role in enhancing repair. Indeed, studies are coming to light that suggest that the extracellular matrix produced by these contaminating fibroblast-like cells may also have an important role in mediating repair [29–31]. For this reason it is quite important to identify the individual cells used in transplant-mediate repair to assess their contribution to resulting regeneration. To date there has been no markers identified that specifically labels OECs or distinguishes them from Schwann cells. Recently it has been proposed that calponin is a specific marker which is able to distinguish OECs from Schwann cells [11, 24]. Using this marker to immunolabel cultures of OECs, a mixture of p75NTR positive/calponin positive and p75NTR positive/calponin negative cells were seen and it was proposed that cells lacking calponin immunoreactivity was attributed to contaminating Schwann cells. For this reason the authors suggest that any myelination seen in vivo after OEC transplantation was solely due to contaminating Schwann cells . This hypothesis has been challenged with evidence demonstrating a lack of calponin staining in the adult rat olfactory system . In this report only adult tissue was examined as it was suggested autologous transplantation is likely to be the basis of clinical translation studies, but an ongoing clinical trial in China is using embryonic olfactory tissue as their source of tissue . Furthermore, it is necessary to examine calponin expression in neonatal OECs and embryonic tissue to allow comparison with the many cell transplantation studies in the literature that has used neonatal OECs and with the original proteomic analysis which used foetal OECs . A study on neonatal and embryonic tissue allows comparison to be made of calponin expression during development and any possible age-related expression in OECs.
In our study we demonstrate that calponin immunoreactivity may be detected in both OECs and Schwann cells in culture although the expression is not typical of calponin immunoreactivity reported in the literature. The calponin immunoreactivity for the glial cells was punctate and nuclear and not fibrillar as seen in muscle cells  or in fibroblast as demonstrated in this study (Figure 5) and by others . However, omitting the primary antibody reduced but did not remove this punctate staining around the nucleus. On the other hand, we found similar nuclear, punctate immunoreactivity in strongly positive fibroblasts which suggests the punctate staining seen in Schwann cells and OECs is more likely to be background non-specific staining. We initially hypothesized that the nuclear staining detected in the OECs and Schwann cells may be due to proteinase K treatments (recommended for tissue immunocytochemistry by the antibody supplier) but we had identical staining in the presence and absence of proteinase K with methanol fixation. In support of this, all of our immunohistochemistry of tissue sections was carried out with proteinase K pretreatment, but still did not reveal calponin staining in OECs. Thus, our data using neonatal and embryonic tissue support the results from adult olfactory tissue that calponin is not a marker for OECs .
Our in vivo studies identified strong calponin immunoreactivity in the connective tissue of the LP of neonatal rats. Heterogeneous antigenic expression was detected in tissue sections and purified cultured cells of the classical connective tissue markers, eg. antibodies to fibronectin, α-SMA, [34–36] and Thy1.1  (for fibroblasts and muscle cells), together with the calponin antibody. Section of neonatal mucosa showed regional differences in connective tissue markers suggesting that subpopulations of fibroblasts exist in the LP. The middle section of the LP contained α-SMA immunoreactivity while the area adjacent to the OE expressed mainly fibronectin and Thy1.1. A proportion of this α-SMA immunoreactivity could be attributed to smooth muscle cells lining blood vessels but the distribution of α-SMA immunoreactivity was too large to be due to these cells alone. In cell cultures prepared from the olfactory bulb and sciatic nerve these markers identified heterogeneously labeled fibroblast-like cells. This is consistent with reports that α-SMA, calponin and Thy1.1 characterize subpopulations of fibroblasts [34, 36, 37]. Thus, our data suggest that connective tissue from the olfactory mucosa is comprised of several subclasses of fibroblasts. Although fibronectin labels connective tissue we have seen coexpression with p75NTR confirming reports that OECs express fibronectin  and that fibronectin expressed in the olfactory nerve layer of the olfactory bulb is likely to be due to OECs . However, it has been suggested that neural crest precursor cells can generate Schwann cells and endoneurial fibroblasts raising the possibility that this type of cell may exist in the developing olfactory mucosa which transiently coexpress p75NTR and fibroblast markers . Interestingly, the expression of calponin in the connective tissue of neonatal LP may reflect developmental changes, since the olfactory system of embryonic rats lacked calponin immunoreactivity. This finding may have relevance for CNS repair as mixed olfactory tissue is being proposed as a more potent reparative cell mix for transplant-mediated repair of spinal cord injury [29–31]. To be able to identify and work with these different cells types may lead to a better understanding of their role in CNS repair.
It has also been suggested that preparations of cultured OECs are contaminated with Schwann cells . OECs used in this study are prepared from neonatal olfactory bulbs using the O4 antibody and fluorescence activated cell sorting  and are over 98% p75NTR pure. Although we have seen lack of calponin staining in either of our Schwann cells or OECs preparations, we believe that our OEC preparations are devoid of Schwann cells due to their inability to form any boundary in confrontation assays [17, 20]. To support this data we carried out confrontation assays with OECs/astrocytes and Schwann cells/astrocytes and immunolabeled the cells with calponin and GFAP. Once more we saw the differential migratory behavior of OECs and Schwann cells when in contact with astrocytes and unexpectedly calponin immunoreactivity was associated with astrocytes. Immunolabeling pure cultures of astrocytes from two different sources supported the finding that astrocytes express calponin. Other reports have demonstrated calponin expression in the brain associated with hippocampal and cerebellar neurons as well as radial glia, Bergman glia, glia limitans and mature astrocytes [41, 42]. However these studies used an antibody to the acidic isoform of calponin. Calponin is developmentally expressed as 3 isoforms; α,β and γ. The basic α and β isoforms have molecular weights of 34 kD and 29 kD respectively. An acidic isoform of calponin has also been reported with a molecular weight of 36 kD . It is this acidic isoform that has been reported to be expressed in a range of tissue and not just muscle cells [35, 42]. The antibody we used in this study is the same antibody described by Rizek and Kawaja  and the same clone (different supplier) as described by Ibanez et al.,  which recognizes the basic calponin isoform. This suggests the basic calponin isoform can be expressed by astrocytes in vitro. Calponin expression in these astrocytes was heterogeneous and may reflect subsets of astrocytes or a differential expression during the cell cycle [44, 45].