Introduction
For over two decades researchers have been studying the therapeutic potential of olfactory ensheathing cells (OECs) to treat traumatic injuries to the central nervous system (Barnett and Riddell
2007; Mackay-Sim and St John
2011; Raisman et al.
2012; Gladwin and Choi
2015). OECs are unique glial cells that exist exclusively within the olfactory system and perform a vital role in supporting the continual turnover of short-lived olfactory receptor neurons (ORNs) throughout adult life (Doucette
1993). New ORNs must accurately extend their axons from their origin in the olfactory epithelium, along the peripheral olfactory nerves, and into the central nervous system where they must form appropriate connections within the olfactory nerve layer of the olfactory bulb. The olfactory system is the only location in humans where such axon growth and path-finding occurs continuously across the peripheral-central nervous system boundary throughout life (Doucette
1991). The ensheathment of ORN axons by processes of OECs along the entire olfactory pathway is vital for this to occur. OECs are dynamic and versatile glia uniquely possessing characteristics of both peripheral Schwann cells and central nervous system astrocytes. These properties enable OECs to maintain both the functioning of mature ORNs and promote and support new ORN growth in both the peripheral and central nervous system.
With this exceptional combination of properties, OECs are prime candidates for therapeutic transplants to promote CNS regeneration. To date, hundreds of studies in experimental nervous system injury models and recent studies in humans have provided convincing evidence to support the clinical development of OEC transplantation (Mackay-Sim et al.
2008; Granger et al.
2012; Tabakow et al.
2014; Watzlawick et al.
2016). These studies have shown OECs can provide a wide range of therapeutic effects. OEC transplants have been reported to enhance tissue preservation and promote functional recovery by; reducing inflammation and lesion cavitation; promoting neuronal survival and plasticity; promoting angiogenesis; enhancing remyelination of spared axons, and promoting regeneration of damaged axonal pathways by bridging the lesion with a growth-permissive substrate (Keyvan-Fouladi et al.
2003; Lakatos et al.
2003; Khankan et al.
2016). A recent meta-analysis of 62 experimental studies in rodent models of SCI concluded that OEC transplants enhanced functional recovery with an effect size of 19.2 % (after adjusting for publication bias and missing data) measured using the Basso, Beattie, and Bresnahan locomotor scoring method (Watzlawick et al.
2016).
In experimental transplantation studies OECs are most often obtained from the olfactory nerve layer (ONL) of the olfactory bulb (OB), and less commonly from the olfactory mucosa. OECs can be isolated from adult or embryonic/foetal tissue and expanded to a limited degree in culture. Commonly, OEC transplants contain a mixture of cells depending on the tissue source, however despite the potential complexity of the cell mixture, often only details of the OEC and olfactory fibroblast components are described in published papers. The majority of our knowledge of OEC biology and reparative properties has arisen from rodent studies. In particular, the immunocytochemical characteristics of OECs have been well studied in rats and mice with the general consensus that OECs express the low affinity neurotrophin receptor P75
NTR, S100 and GFAP in situ and in vitro (Gong et al.
1994; Franceschini and Barnett
1996; Au et al.
2002). These three antigens are the most commonly used markers to identify OECs in therapeutic cultures (Kawaja et al.
2009).
To improve culture yields, rodent OECs can be purified from mixed cell cultures or enriched using various methods, most commonly by cell sorting using the expression of P75
NTR. This can be performed on the initial cell suspension obtained after tissue digestion, or after plating and culturing for several days (Kawaja et al.
2009). One drawback to this method is that contaminating Schwann cells present in peripheral nerve bundles in the mucosa and surrounding arterioles in the ONL also express P75
NTR and so, may be co-isolated along with OECs. Due to similarities in their antigenic characteristics, currently there are no established protocols for purifying rodent OECs from Schwann cells. Studies in larger animal models have revealed inter-species differences in the antigenic characteristics of OECs in situ (Wewetzer et al.
2011). In a canine model, P75
NTR was expressed in non-myelinating Schwann cells and in connective tissue surrounding olfactory nerves in situ, but not in OECs (Bock et al.
2007). As canine OECs only upregulate P75
NTR after a period of time in culture, Schwann cells can be removed from the initial cell digest using P75
NTR immunopanning (Ziege et al.
2013). The resulting suspension can be cultured to allow time for OECs to upregulate P75
NTR and then subjected to a second round of immunopanning to isolate OECs from any remaining contaminating cells. These studies highlight the importance of corroborating OEC markers when extrapolating protocols developed in rodent models to other species. A lack of P75
NTR expression in OECs in situ was also reported in cats and guinea pig (Smithson and Kawaja
2009). Immunocytochemical characterisation in situ not only provides markers for purification and identification of OECs, but also provides insights into their complex biology. Studies in rodents have demonstrated OECs differentially express several antigens in the olfactory bulb and mucosa, and between the inner and outer layers of the olfactory bulb (Vincent et al.
2005). OECs in the olfactory nerves and outer ONL express P75
NTR, whilst OECs in the inner ONL do not express P75
NTR but do express neuropeptide Y and GFAP (Franceschini and Barnett
1996; Au et al.
2002). These data suggest functional differences may exist between anatomical OEC populations. Indeed, studies have shown OECs from different regions of the ONL and olfactory nerve differ in their phagocytic capabilities, migratory and cell–cell interactive behaviours (Lohr et al.
2014; Ekberg and St John
2015). These differential properties could be exploited to enhance the efficacy of therapeutic transplants.
The immunocytochemical characterisation of human OECs in situ has generally been limited to the analysis of biopsies of the adult human olfactory mucosa; very little is known about the antigenic characteristics of adult and developing human OECs within the olfactory bulb and nerve roots. The small number of studies that have described human OECs in situ in the adult olfactory mucosa have presented conflicting results with some suggesting OECs do express P75
NTR (Bianco et al.
2004; Kachramanoglou et al.
2013; Winstead et al.
2014), whilst Holbrook et al. (
2016) suggest that similar to canines, olfactory perineurial cells express P75
NTR, but OECs do not (Holbrook et al.
2016). Validation of P75
NTR expression or lack thereof in human OECs in situ is necessary for developing protocols for correctly identifying and enriching OECs in cultures for future clinical trials.
For clinical application, autologous OEC transplants would be ideal however, obtaining OECs from the olfactory nerve layer of a patient requires an invasive and risky brain operation resulting in the unilateral loss of an entire olfactory bulb and risks of anosmia, meningitis, stroke or death. Biopsies of olfactory mucosa are a safer alternative but isolating OECs from these biopsies can be difficult and yields can be variable, especially where a patient may possess a pre-existing nasal disease (Kachramanoglou et al.
2013). Alternatively, in the future OECs may be generated from patient stem cells such as hair follicle neural crest stem cells or from induced pluripotent stem cells. However, despite some recent progress, our knowledge of the pathways that control OEC development from the neural crest, and how these pathways differ from Schwann cell development are limited (Barraud et al.
2010; Miller et al.
2016). Thus, additional allogeneic sources of OECs may be required until appropriate differentiation protocols to generate OECs from patient stem cells have been developed and validated.
Despite ethical considerations, discarded human foetuses could provide an additional source of human olfactory tissue for research purposes and a supplemental cell source during the initial clinical development of OEC therapies. Transplants of human foetal cells have been used historically to provide proof-of-concept for various regenerative cell therapies such as photoreceptor replacement therapy (Humayun et al.
2000). Foetal OECs could be used clinically where it is either not possible to obtain patient autologous OECs due to pre-existing pathologies or where patient autologous OEC cultures are not of sufficient yield or quality. Proof-of-concept studies demonstrating the feasibility of allogeneic OEC grafts with short-term immunosuppression in animal models of spinal cord injury have already shown some promising results including axon regeneration and recovery of directed forepaw reaching (Li et al.
2016).
To address the knowledge gap regarding the characteristics of human OECs in situ and to corroborate expression of rodent OEC markers in human foetal OECs, we undertook an immunocytochemical analysis of the 11–19 pcw human foetal olfactory system.
Discussion
The immunocytochemical characterisation of OECs in the olfactory system of animal models such as rats and dogs has enhanced our understanding of OEC physiology and established a range of antigenic markers that can be used to identify OECs in experimental cultures. Identification of surface markers such as P75NTR has also enabled the development of important purification techniques to enhance culture yields and eliminate unwanted cell types. In contrast, limited information is available regarding the antigenic characteristics of OECs in situ in the adult and developing human olfactory bulb and olfactory nerves. Studies in various animal models have shown slight but important differences in the characteristics of OECs between species. Demonstrating the identity and purity of cell transplants is a prerequisite to gaining authorisation for clinical trials of advanced therapeutic medicinal products. Thus, a thorough characterisation of human OECs in situ would be highly beneficial for the clinical development of OEC transplants. To address this, we investigated the in situ expression of common rodent OEC markers in the developing 11-19 pcw human foetal olfactory system. We assessed immunoreactivity to S100, P75NTR, PSA-NCAM, Gap-43, vimentin, nestin, NPY, GFAP, and O4, and to a lesser-known marker for OECs: SOX10.
Our results indicate that the antigenic properties of OECs in the developing human have many similarities but also some important differences with rodent OECs in situ. Franceschini and Barnett (
1996) provide a detailed description of the antigenic characteristics of the developing and postnatal rat olfactory system (Franceschini and Barnett
1996). From E14-P0, the olfactory nerves were described as consistently PSA-NCAM+ on the inside surrounded by a thin filamentous P75
NTR+ outer layer. At the ventral surface of the OB, PSA-NCAM expressing cells extended through openings in a P75
NTR+ layer that wrapped around the PSA-NCAM+ tissue. PSA-NCAM immunoreactivity was restricted to the outer ONL until birth when it was also detected at the ONL/GL boundary. This configuration of an external P75
NTR layer surrounding a PSA-NCAM+ layer was observed along the entire olfactory pathway. They concluded that the PSA-NCAM+ tissue represented astrocyte-like OECs enwrapped by the P75
NTR+ Schwann-like OECs. An additional layer of P75
NTR immunoreactivity covering the OB and entire cortex was also observed and identified as meningeal tissue. The migratory mass and olfactory axons extending from the OE to the OB consistently labelled with NCAM, vimentin and GAP-43 throughout development.
Our results showed some similarities with the configurations of immunoreactivity described by Franceschini and Barnett (
1996). We observed consistent immunolabelling with vimentin, S100, NCAM and GAP-43 along the entire length of the ONs from the OE to the OB in our human foetal samples. Furthermore, we also observed P75
NTR in a meningeal layer covering the OB, and in similar configurations with PSA-NCAM immunolabelling. Consecutive double-labelled sections revealed that PSA-NCAM+ tissue occupied areas surrounded by P75
NTR+ tissue in the ONs, similar to that described by Franceschini and Barnett. However, our results suggest a different cellular origin of these two antigens in the developing human foetus from that proposed by Franceschini and Barnett. Our high magnification confocal fluorescent micrographs of the olfactory nerves revealed three layers: S100− vimentin+ P75
NTR+ ONFs surrounding S100+, SOX10+ and vimentin+ OECs which ensheathed NCAM, GAP-43, TUJ and PSA-NCAM+ bundles of ORNs axons.
We identified OECs by robust S100 and SOX10 immunolabelling that was consistently observed in cells with the morphology of OECs that tightly ensheathed TUJ+ ORN axons in olfactory nerves, at all foetal ages studied. S100/SOX10 expression in OECs was consistent along the entire olfactory pathway and throughout the ONL and was thus used where possible in triple antibody labelled sections as a marker for OECs. We also confirmed NCAM and GAP-43 as markers of ORN axons and not OECs in olfactory nerves by co-labelling with TUJ (ORNs axons) and SOX10 (OECs). Our results were consistent with previous studies showing NCAM and GAP-43 in ORN axons in human and mouse (Chuah and Au
1992; Au et al.
2002; Iwema and Schwob
2003; Maresh et al.
2008). Co-labelling with these markers revealed that PSA-NCAM was present in ORN axons but not obviously in OECs. It is possible that PSA-NCAM was present on OEC surfaces in contact with ORN axons but this was not distinguishable by fluorescence microscopy. Nevertheless, the pattern of PSA-NCAM immunoreactivity we observed was consistent with previous reports that only subpopulations of immature ORNs express this antigen (Miragall and Dermietzel
1992).
Our most significant finding was that human OECs do not appear to express P75
NTR but perineural ONFs surrounding OECs in olfactory nerve fascicles do strongly express P75
NTR. This was consistent among all the samples analysed and throughout the olfactory system in both the ONs and outer ONL. Expression of P75
NTR by ONFs and not OECs was most obvious in precise cross-sections and z-stack confocal images of olfactory nerves. Co-labelling with S100 and SOX10 demonstrated that P75
NTR was on cells surrounding OECs and did not appear to be on OECs themselves. Without co-labelling with S100, the similar distribution and morphology of P75
NTR immunolabelling in the olfactory nerves could be mistaken for labelling in OECs. These results are similar to those found in adult canine, guinea pig and cat, but in contrast to rat and murine OECs (Gong et al.
1994; Franceschini and Barnett
1996; Au et al.
2002; Au and Roskams
2003; Bock et al.
2007; Smithson and Kawaja
2009). These results are also in contrast with Liu et al. (
2010) who observed substantial P75
NTR immunoreactivity in the ONL and surrounding glomeruli in the olfactory bulb in 20 pcw human foetuses (Liu et al.
2010). Differences between our study and Liu et al. could be due to differing antibodies or foetal sample age. Strong immunostaining in peripheral nerves in control tissue sections taken from the same foetal samples, and in the meningeal tissue (which is well reported) provides confidence that the P75
NTR antibody used in this study was effective.
Electron micrograph studies have shown the configuration of OEC ensheathment of olfactory axons and their compartmentalisation into fascicles by basal lamina and ONFs differs among species (Herrera et al.
2005; Kawaja et al.
2009; Smithson and Kawaja
2009). In rodents, spaces between OEC-axon units are packed with collagen fibres and only a single loose layer of ONFs surrounds the outside of ONs (Field et al.
2003). In larger mammals such as cats and non-human primates
Macaca fascicularis, each OEC-axon unit within a larger fascicle is separated by collagen fibres and a thin single cell layer endoneurium-like fibroblastic process lacking a basal lamina (Herrera et al.
2005). Multiple fascicles are then surrounded by a second layer of perineurial ONFs. Our results suggest the configuration in humans is similar to
Macaca fascicularis. The P75
NTR+ layers we observed surrounding the S100/SOX10+ OEC-axon units and fascicles are these endoneurial and perineurial ONFs. Smithson et al. (2009) and Bock et al. (
2007) reported a similar configuration of P75
NTR+ cells surrounding olfactory nerves in adult guinea pigs, cats and dogs and concluded these cells were ONFs. They did not observe P75
NTR in OECs in these species (Bock et al.
2007; Smithson and Kawaja
2009).
Our tissue sections of the foetal olfactory system often contained nerves of peripheral origin coursing through the nasal mucosa and alongside blood vessels in both the peripheral and intracranial compartments of the olfactory system. These small vessels were present within the outer and inner ONL and along the surface of the olfactory bulb. Similar to olfactory nerves we observed P75NTR immunolabelling in perineurial fibroblasts surrounding peripheral nerves; however, in contrast to OECs, Schwann cells in these nerves also strongly expressed P75NTR determined by clear overlap with S100 immunoreactivity. Schwann cells also expressed significantly more GFAP compared to OECs. Similar to that described in the developing rodent olfactory system, GFAP expression in OECs in the olfactory nerves and ONL was very sparse whilst in peripheral nerves, GFAP was intense and abundant throughout the entire nerve bundle. These data show clear differences in the phenotype of Schwann cells and OECs during the developmental ages studied.
The finding that P75
NTR is expressed by Schwann cells and ONFs but not by OECs in situ has implications for developing techniques to purify, identify and culture human OECs. Traditional methods developed in rodents whereby OECs are isolated by P75
NTR immunopanning the initial cell suspension would result in a culture that did not contain OECs, only Schwann cells and ONFs. Alternatively, P75
NTR immunopanning after a period in culture would allow time for OECs to upregulate P75
NTR, but would also isolate Schwann cells and presumably ONFs. Using both these protocols sequentially to first remove Schwann cells and ONFs, and then after a culture period to isolate OECs could provide highly pure OEC cultures similar to protocols described for canine OECs (Bock et al.
2007).
Transplants of OECs generated from the digestion of whole human foetal olfactory bulbs are already being used to treat hundreds of patients for various ailments including SCI, stroke, cerebral palsy and neurodegenerative diseases such as ALS (Huang et al.
2006,
2009; Chen et al.
2012,
2013,
2014; Xi et al.
2013). The transplanted cells in these studies have been described as OECs and characterised using antibodies towards P75
NTR. No selective purification methods have been described to isolate OECs from other cells of the olfactory bulb indicating these cultures are likely to contain a wide range of cells and not just OECs. Liu et al. (
2010) characterised human foetal OB cultures generated using similar methods and found them to contain just 23 % P75
NTR+ cells. Other cells in the cultures were GFAP+, S100+ and nestin+, possibly neural stem and precursor cells, reactive astrocytes or microglia (Liu et al.
2010). Another report by Guest et al. (
2006) found similar cultures described as OECs contained nestin and GFAP but no S100 and were morphologically reminiscent of human neurosphere cultures; the transplants were not clearly OECs (Guest et al.
2006). Outcomes from these human foetal OEC clinical studies have been controversial. The ability to correctly identify and purify human OECs is vital to understanding and developing effective OEC therapies. We did not observed nestin in OECs in the ONL of our foetal OB cryosections however, many other cells that were not OECs in the glomerular and deeper OB layers did express nestin, suggesting this marker is not useful for identifying human OECs generated from OB tissue.
SOX10 is a transcription factor required for the differentiation of Schwann cells and for the development of OECs in mice and chick (Kelsh
2006; Barraud et al.
2010; Forni and Wray
2012; Pingault et al.
2013). Elucidating the developmental pathways of OECs will be essential for developing differentiation protocols to produce stem cell derived human OECs. SOX10 has also been observed in OECs in the mucosal olfactory nerves of a single 8-week human foetus (Pingault et al.
2013). Our observations of SOX10 confirm the presence of this key transcription factor in OECs throughout the ONs and ONL in the 11–19 pcw developing human.
Often, OECs are described as several different populations in vivo depending on their expression of various markers. Franceschini and Barnett (
1996) described two OEC populations in rodent: astrocyte-like expressing PSA-NCAM and fibrous GFAP, and Schwann-like expressing P75
NTR with diffuse GFAP (Franceschini and Barnett
1996). In the olfactory bulb of mice, Au et al. (
2002) identified P75
NTR− S100+ NPY+ OECs in the inner ONL and P75
NTR/PSA-NCAM+ OECs in outer ONL (Au et al.
2002). Pixley (
1992) also described Schwann-like (S100/P75
NTR/GFAP+) and astrocyte-like (GFAP+ and S100/P75
NTR−) OECs in situ in the olfactory nerves of neonatal rats. Intensely GFAP+ astrocyte-like OECs occurred as isolated cells within the ONs, orientated parallel with the longitudinal axis of the nerve (Pixley
1992). We observed one population of OECs within both the ONs and ONL of the developing human olfactory system that expressed S100, SOX10, vimentin and heterogeneously small amounts of fibrous GFAP (Table
3). We were unable to confirm S100 co-labelling in GFAP+ OECs as both antibodies were raised in rabbit and our anti-mouse GFAP only detected GFAP in small populations of astrocytes in the forebrain. The GFAP+ OECs we observed were similar to that described by Pixley (
1992) occurring as isolated cells orientated parallel with the nerve (Fig.
4d). These cells may represent a second OEC population, the astrocyte-like OECs. We also describe another cell type that we assumed to be ONFs based on its anatomical morphology i.e. ensheathing S100+ OECs and not TUJ ORN axons. This cell type was S100− vimentin+ GFAP− P75
NTR+, some of which also expressed nestin.
Table 3
Summary of the immunohistochemical analysis of the human foetal olfactory system
Olfactory nerve | +++§§§ | +++§§§ | +++§§§ | A | ++ ± to § | +++§§§ | +++§§§ | A to +++ §§§ | +++§§§ | +++§§§ | +§ | ++§§ | A |
Olfactory nerve layer (outer/inner 17 pcw only) | +++§§§ | +++§§§ | +++§§§ | A | +§ | +++ § (+++§/+++±) | +++§§§ | A to ++ § | +++§§§ | +++§§§ | ++§ | A (+ ±/A) | A |
Glomerular layer (17 pcw only) | ++§§§ | +++§§§ | A | A | +§ | A | A | A | +++§§§ | ++§§ | ++§§ | A | A |
Olfactory epithelia | +§§ | +++§§§ | ++ ± | A | A | A | +§§ | ++ § | +++§§§ | ++§§ | A | A | A |
Co-label with p75? | ND | No | In peripheral nerves. | ND | In peripheral nerves. | NA | Yes - In Schwann cells and rarely in olfactory perineural cells. | ND | ND | ND-but labelling of adjacent tissue sections suggests yes. | ND-but labelling of adjacent tissue sections suggests yes. | ND | ND |
Clearly expressed by OECs? | No | No | Yes, extensively. | No | Yes, in small amounts. | No | Yes, extensively. | No | No | Yes, extensively. | No | ONL: No ON:Unsure | No |
We did not observe any differences in the immunolabelling characteristics of OECs between the ONs and ONL other than O4, which labelled ONs but was absent in the ONL. O4 was previously considered to be a surface marker for rodent OECs and has been used by several groups to enrich OEC cultures using FACs (Franceschini and Barnett
1996). O4 immunoreactivity on rodent OECs in vivo was initially described by immunofluorescence. Later studies utilising immunoelectron microscopy revealed this antigen was actually present on ORN axons and not OECs (Wewetzer et al.
2005). This study concluded that O4 observed on OECs in vitro was from phagocytosed axonal fragments and not endogenous expression; this has been disputed and remains unresolved. We were unable to identify the origin of O4 immunoreactivity in the human foetal olfactory system and thus, this requires further investigation by immunoelectron microscopy. We were also unable to identify a NPY+ OEC population equivalent to that described by Ubink in the ONL of embryonic rats suggesting this neurotrophic factor may not be involved in human olfactory development at the foetal ages studied (Ubink and Hökfelt
2000).
Antigenic properties of ONFs
The identification of P75
NTR+ S100− ONFs in this study raises a number of important questions: Do we see similar P75
NTR immunoreactivity in adult human ONFs, alluded to by Holbrook et al.
2016? What are the morphological and antigenic characteristics of these cells in culture? Do they continue to express P75
NTR in vitro and if so, what proportion of these cells persist in OEC cultures that are traditionally characterised based on P75
NTR purity. In rodent OEC cultures, ONFs are normally characterised by positive labelling for fibronectin, Thy-1, Smooth muscle actin and negative for P75
NTR (Li et al.
2008; Toft et al.
2013). This is similar to the phenotype of endoneurial fibroblasts found in the peripheral nerves of mice that along with Schwann cells differentiate from a non-neuronal restricted progenitor cell originating from the neural crest (Joseph et al.
2004). This non-neuronal restricted progenitor cell is characterised by expression of P75
NTR, S100b and desert hedgehog. Differentiation along the fibroblast lineage results in the loss of P75
NTR and desert hedgehog and expression of Thy-1. Human ONFs clearly differ antigenically from rodent ONFs and peripheral nerve endoneurial fibroblasts. Dissecting the function of P75
NTR expression is difficult as this pan-neurotrophin receptor and NOGO-66 coreceptor mediates a wide range of extremely complex signals, many of which are still being deciphered. P75
NTR is expressed in neurons and glial cells as well as many mature non-neuronal cells such as endothelial cells and perivascular fibroblasts. It is also expressed in various types of mesenchymal cells and embryonic and adult stem cells. Among many functions, P75
NTR mediates cell death and survival, proliferation, differentiation, migration, and has essential roles in controlling the development of the nervous system including neuronal survival, neurite outgrowth and retraction, and myelination (Rabizadeh and Bredesen
2003). Schwann cell P75
NTR is vital for promoting peripheral nerve regeneration after injury (Zhou and Li
2007). P75
NTR is also involved in controlling pluripotency and modulating cell-fate decisions in stem cells (Tomellini et al.
2014). The significance of P75
NTR in human ONFs remains to be investigated.
As well as P75
NTR, our results indicate that a proportion of human ONFs also express nestin. Nestin is a cytoskeletal intermediate filament protein associated with an immature phenotype expressed abundantly in progenitor and multipotent stem cells and in various tissues during development, particularly in the nervous system (Michalczyk and Ziman
2005). During differentiation nestin is normally replaced by cell type specific intermediate filament proteins but can be re-expressed transiently during tissue regeneration and wound healing. The expression of nestin in human ONFs is not entirely unexpected as nestin has been previously reported in myofibroblasts in various different tissues (Kishaba et al.
2010), in fibroblasts cultured from human olfactory mucosa biopsies (Wu et al.
2013), and in a sub-population of endoneurial fibroblast-like cells in rodent and human peripheral nerves (Richard et al.
2014). Richard et al. (
2014) reported that after peripheral nerve injury in rodents, the number of nestin expressing endoneurial fibroblast-like cells significantly increased leading them to speculate that these cells may have progenitor-like properties. Similarly, the presence of nestin in human ONFs could indicate progenitor-like properties. It is currently unknown to what extent human ONFs are needed in OEC transplants to achieve therapeutic outcomes. Previous studies in rodents have suggested that ONFs are a crucial component of therapeutic OEC transplants facilitating the formation of OEC bridges through which axons can regenerate across growth inhibitory CNS tissue. Electron microscopy studies have shown that after transplantation into spinal cord lesions, ONFs wrap around OEC channels forming a sheath similar to their physiology in situ (Li et al.
1998
). During culture, ONFs also contribute to the production of a gel-like endogenous matrix that provides a useful vehicle for transplanting OECs to the injury site. Thus, although P75
NTR immunopanning as described may allow the generation of human OECs cultures free from Schwann cells, further surface markers will be needed to isolate ONFs from Schwann cells to investigate their role in the regenerative properties of OEC transplants.
In summary, we show that human foetal OECs possess both similarities and important differences with rodent OECs in situ. Most significant of these findings is that ONFs and not OECs express P75NTR in the developing foetus. However, this antigen could still be used in protocols to remove P75NTR+ Schwann cells and fibroblasts and eventually isolate OECs after P75NTR upregulation in vitro, to generate purified cultures from foetal tissue. We define OECs throughout the 11–19 pcw human olfactory system as S100/vimentin/SOX10+ with very small amounts of GFAP. Expression of O4 on OECs and not ORN axons in situ remains to be proven and presently it is not a robust surface marker for OEC purification. PSA-NCAM is also not a suitable surface marker for OEC purification owing to its heterogeneous expression on ORN axons and lack of reliable labelling of OECs in the human foetus. Our results highlight the importance of corroborating cell markers when translating cell therapies from animal models to the clinic.