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1. Introduction
Neurons are one among the very few differentiated cell types which are strictly post-mitotic and therefore non-renewable by sister cell divisions. The production of new neurons, or neurogenesis, does though occur in limited areas of adult mammalian nervous system, due to the existence of intrinsic stem cells which has been formally demonstrated by a primary culture approach on adult brain tissues from mouse [1], man [2,3] and other species [4]. These “neural stem cells” satisfy the general stemness criteria, i.e.: illimited proliferative activity, self-renewal capacity, multipotency (i.e. capacity to generate all the differentiated cell types of their host tissue, which for neural stem cells encompasses neurons, astrocytes, oligodendrocytes), proliferative reactivity to tissue lesions and physiological signals [5]. This discovery has raised great promise for clinical repair in neurology and led to coin a novel principle in cell therapy, which consists in replacing lost neurons by grafting
2. General properties of neural stem cells in adult mammals
Neural stem cells are identified and isolated in primary cultures of adult nervous tissues in the presence of epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF), by the growth of cellular masses called “neurospheres” which include the three neural lineages: neurons, astrocytes, oligodendrocytes [1]. This primary culture is termed “neurosphere assay” (NSA) and allows to generate and expand great numbers of proliferative neurogenic cells through successive passages [11]. Recent methodological refinements allowed to isolate and distinguish neural stem cells
Beside the two initially recognized and most active neurogenic structures of adult mammalian brain (the subventricular zone of telencephalon and the subgranular layer of hippocampal dentate gyrus), several topographically restricted regions of the central nervous system were shown to yield neurospheres in EGF-bFGF-treated primary culture and therefore to contain neural stem cells [13,14]. Spatial frequency among tissue and
Neural stem cells generate differentiated progenies, like other types of adult stem cells, through several intermediate stages which can now be distinguished by their respective marker combinations – or “molecular profiles” or “transcriptomic signatures” (Figure 1). The biological properties of neural stem cells from adult nervous tissues are reminiscent of those characterizing neural progenitors from embryos, especially
Figure 1.
The “neuropoietic system” of adult neurogenesis. Modelized from references 15,38.
3. Neurosphere isolation from adult pig brain subventricular zone
Neural progenitors have been isolated from foetal porcine brain by several laboratories in the last years, by using the neurosphere assay. We decided to develop isolation of adult brain-derived neural stem cells, because of their higher safety than other available proliferative neurogenic cell types in terms of oncogenic potential (cf above). Neural stem cells are found only in rare and discrete structures, the best characterized and most productive of which is the subventricular zone (SVZ) of the forebrain in rodents. We have isolated and characterized for the first time the neural stem cells of adult pig SVZ, by transposing the “neurosphere assay” from routine protocols of rodent studies [26].
3.1. The key to success for the “neurosphere assay”: Adequate tissue sampling
This particular primary culture is known to be hampered by two pitfalls: i) delay between
In order to minimize the duration of brain tissue sampling, which is lengthened in adult pig by the requirement of dorsal skull sawing, we performed the surgical approach of telencephalic subventricular region in deeply anesthetized pig instead of immediately after sacrifice. A rough 10mm-thick, transverse section of midbrain was explanted and immediately transferred into a large Petri dish filled with fresh Hank’s medium, and the subventricular zone was then microdissected out of this thick slice on the basis of macroscopical neuroanatomical landmarks by reference to a detailed pig brain atlas [27]. Despite this precaution, our first neurosphere assay attempts on adult pig SVZ failed, which was subsequently attributed to improper tissue sampling. Indeed, the telencephalic subventricular zone is proportionally much larger in adult pig than in adult rodents, and proliferative activity is already known as highly heterogeneous among the whole anatomical extent of SVZ in rodent brain [28]. We performed therefore the subregional mapping of proliferative activity within adult pig SVZ, by Ki-67 immunohistochemistry using a routine protocol [29,30]. The Ki-67 protein is indeed a phylogenetically conserved marker which is expressed exclusively during the cell division cycle of eukaryotic cells, but throughout all its successive phases (G1-S-G2-M); it is therefore widely used in clinical anatomo-pathology and oncology. Proliferative cells, appearing
Figure 2.
3.2. Primary neurospheres from adult pig brain as a vehicle of neural stem cells
For primary cell culture (“neurosphere assay”), a piece of SVZ tissue was microdissected from the pig forebrain slice in low-calcium artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 5 mM KCl, 3.2 mM MgCl2, 0.1 mM CaCl2, 26 mM NaHCO3, 100 mM glucose, pH 7.38, according to Weiss et al., 1996). As previously described [30], the tissue sample was rinsed twice with aCSF, and digested first in 40 U cystein-EDTA-βmercaptoethanol-preactivated papain (Sigma, L’Isle D’Abeau, France) for 10 min at 37°C, then in 250 µL undiluted TrypLETM Express solution (heat-resistant, microbially-produced, purified trypsin-like enzyme, Gibco cat 12604-013, Invitrogen, Cergy-Pontoise, France) for 10 min at 37°C. After the addition of 750 µL of fresh aCSF and centrifugation for 8 min at 400g at room temperature, the cell pellet was resuspended in 1mL culture medium (Dulbecco’s modified Eagle medium [DMEM, Sigma], B27 supplement [Gibco Invitrogen], 200 U/mL penicillin and 200 µg/mL streptomycin [Gibco Invitrogen]) containing 20ng/mL Epidermal Growth Factor (EGF, Gibco Invitrogen) and 20 ng/mL basic Fibroblast Growth Factor (bFGF, Gibco Invitrogen). The cells were dissociated gently with a 26G steel needle mounted on a disposable 1mL syringe, counted on a Malassez slide, and seeded at various densities according to purported utilization. Optimal results were obtained with either Corning 24-well plates or with Falcon 6-well ones. Seedings for basic cell biology kinetics were performed at 3,000 cells per mL of culture medium (subclonal concentration). Maximal neurosphere expansion for transplantation or molecular assays was obtained with seeding at 30,000 cells per mL. Cultures must be monitored daily to follow the morphological growth of the neurospheres; when their diameter reaches 150μm indeed, growth slows down while sphere surface apparently thickens and hardens, and culture proves unable to be expanded any more. Passage (i.e. sphere dissociation, cell resuspension and re-seeding as for primary culture) was performed therefore when the majority of spheres was 100-120µm in diameter.
For passage, the primary spheres were collected in sterile tubes, incubated for 45-60 min at 37°C in 250 µL undiluted TrypLETM Express solution (Gibco Invitrogen) per 6 mL culture-derived pellet, and dissociated gently with a 26G steel syringe; dispersed cells were centrifuged, resuspended in fresh proliferative medium, counted ant their dilution adjusted to be seeded as above.
Both primary and passaged spheres were prone to two kinds of utilization:
for neural stem cell expansion, subsequent passages were run in proliferative conditions, i.e. in the presence of 20ng/mL EGF and 20ng/mL bFGF;
for cell differentiation, spheres were picked with a Pasteur pipette, seeded in wells containing poly-D-lysine (Sigma) precoated sterile coverslips and cultured in the above culture medium (DMEM, B27, penicillin-streptomycin) in the absence of EGF and bFGF for 7-10 days.
All culture media were renewed by the replacement of 500 µL of medium per well every 2-3 days.
We have observed in our primary neurospheres from adult pig SVZ the properties which have been established as the seminal criteria of neural stem cells since their pioneering discovery in adult murine brain [1,11].
3.3. Transcriptomic signature of neural stem cells in adult pig SVZ neurospheres
We have assayed the expression of phenotypic markers by RT-PCR on total RNA from proliferative, undifferentiated SVZ neurospheres of adult pig SVZ, which were collected at the second or third generation as described above. Pig SVZ neurospheres displayed positive expression of RNAs encoding for:
nestin, an intermediate filament which is systematically expressed by neural stem cells and embryonic neural progenitors;
the proliferation marker cyclin D1;
the neural stem cell proteins Sox-2, Hes-1, p75NTR (the low-affinity receptor of neurotrophins), Lhx2;
the neuronal proteins βIII-tubulin, doublecortin (DCX), neuron-specific enolase (NSE), Internexin α, Pbx1, Mash1, NeuroD1;
the oligodendrocyte markers CNPase, Olig-2, PDGF-Rα (Figure 3).
Figure 3.
Transcriptomic profile of proliferative neurospheres from adult pig SVZ (modified from original data of reference 26). Agarose gel electrophoretic analysis of amplicons from RT-PCR on proliferative neurosphere RNA with primers designed on the basis of human and/or pig genomic sequences. Stem cell markers: Nestin, Hes-1, Lhx-2, p75NTR, Sox-2, Cyclin D1, Cyclin D2. Committed progenitor markers: Pbx1, βIII-tubulin, internexin-α, NeuroD, NSE, DCX, Mash-1, CNPase, PDGFR-α. PCR products are shown beside co-electrophoresed commercial DNA ladders.
The astrocyte marker glial fibrillary acidic protein (GFAP) has been detected on differentiated neurospheres from adult pig SVZ by immunocytochemistry using the antibody against cow GFAP (cf above) but its transcript could not be detected in the proliferative neurosphere RNAs (probably due to unknown specificity of porcine gene sequence and primers therefore).
The lack of Olig-1 transcripts is expected at this pro-differentiating stage since this gene is expressed in the late phase of oligodendroglial lineage maturation [32,33]; it is also in keeping with Hes-1 detection in the present proliferative spheres, since Hes-1 overexpression was shown to correlate with Olig-1 down-regulation during differentiation [34,35].
In the persistent absence of any specific marker of either neural stem cell or neural committed progenitors, this series of transcripts fits best with the molecular profile of neural stem/precursor cells which has been characterized in rodents and human [36,37,38]. On these bases, we may assume that the major cell type in our adult pig SVZ-derived neurospheres correspond to neural multipotent progenitors, i.e. a stage of the neural lineage that is slightly more mature than the primitive neural stem cells but equally multipotent [26]. This interpretation is supported by additional RT-PCR data in our neurospheres: the absence of the stemness gene transcripts CD133 and SDF-1 [39], the weakness of nucleostemin expression [40], and the lack of detectable cyclin D2, which was demonstrated to specifically drive neural stem cells into division onset by assaying
The present data can be compared with published molecular profiling of neural progenitor cultures from the pig foetal brain [42,43] or adult retina [44,45]. Among the 13 markers that have been addressed by RT-PCR on mRNA extracts of proliferating neurospheres, nine yielded similar results between foetal and adult SVZ, i.e. either negativity (CD133, SDF-1) or positivity (nestin, Sox-2, Hes-1, Pbx-1, β-III-tubulin, doublecortin, Nogo-A). Two differences were encountered between expanded neural cells from adult and foetal brains. The first of these was the detectable expression of cyclin D2 in foetal, but not adult, pig neurospheres. This indicates that neural stem cell expansion is higher in foetal than in adult neurosphere cultures [42]. Furthermore, the lack of Ki-67 detection in pre-differentiation neurospheres from foetal [42], but not adult (present data) supports the same interpretation, since primitive neural stem cells are much less actively dividing than neural precursors at the immediately subsequent maturation stage [46]. Interestingly, the published molecular profile of
In the future, production of neural stem/progenitor cells from the adult pig by the present procedure can promote cell therapy improvements, first in animal models, but also in clinical attempts to repair neural lesions in the use of adult human. By analogy with other tissues [47,48], adult pig neural precursor cells should be feasible for transplantation into diseased humans without immune rejection [49,50]. The present study thus provides the substratum for an alternative source to human allografts for transplantation in neurodegenerative disorders or injuries, which is now open to experimentation.
3.4. In vitro lineage labelling
In order to track
The easiest and long-established tagging method for living cell tracking after transplantation, is incubation of dividing cells with a labeled analog of DNA biosynthesis precursors: nucleotides. Two tools are available: tritiated thymidine (3H-Thy) and bromo-deoxy-uridine (BrdU), and can be detected
Another, more recent procedure allows true lineage-labeling without fading across successive rows of division: it consists in applying a viral vector of the green fluorescent protein (GFP) gene, either by
Figure 4.
Lineage labelling of neurospheres from adult pig SVZ by
4. Improvement of post-lesional neural outcome with primary pig neurosphere transplantation inside a venous autograft
Cell therapy for the nervous system has encountered a major empirical obstacle: in adult mammalian brain, the neurogenic potential of neural stem cells is inhibited by intrinsic tissue microenvironment except for the very few naturally neurogenic areas, which has been formally demonstrated by heterotopic transplantation studies [56]. To overcome this problem, we developed a novel paradigm in order to improve post-lesion regeneration in peripheral nerves: transplantation of
Bridging a long nerve gap with a homotypic venous graft is already used in human neurology in order to ameliorate post-lesional recovery of peripheral nerves [57,58,59,60], but it was barely explored for neuronal cell therapy applications. The disabilities resulting from peripheral nerve lesions in adults typically display no neuron loss but pathway disruption, upon disappearance of distal axonal segments, which was extensively documented to lead to restricted or aberrant synaptic reconnexion of surviving neurons [61]. We postulated that neural stem cell grafting on the lesion site might overcome this limitation, by generating chains of interconnected neurons that would functionally replace the lost nerve substance more efficiently than existing severed neuronal axons.
To test this hypothesis, we attempted transplantation of the proliferating neurospheres from adult pig subventricular zone (SVZ) which we had characterized above, inside an autologous venous graft, following surgical transsection of nervis cruralis with 30mm-long gap in adult pig [53]. The following section summarizes the hallmarks of this study, methods and results [53].
4.1. The experimental cell therapy paradigm and its functional outcome
Our lesion model was unilateral transsection of nervis cruralis with 30 mm-long gap in adult pig, under anesthesia. This lesion induces a major motor defect, consisting in the loss of the right leg extension over the thigh, which can be quantified by electromyography of the crural-innervated muscle quadriceps. Our experimental cell therapy paradigm consisted in bridging the nerve gap immediately after damage with an autologous venous segment (sampled from vena mammalian externalis) which was sutured at the proximal end over the perinevre of the severed nerve, filled through the opposite end of the venous graft with freshly prepared suspension of
Figure 5.
Surgical paradigm for adult pig neural stem cell (ANSCs) transplantation inside a venous graft (modelized from reference
Lesion-induced loss of leg extension on the thigh became definitive in controls (up to 8 months after lesion) but was reversed by 90-180 days after neurosphere-filled vein grafting. Prior to surgery, electromyograms of muscle quadriceps vast internal displayed amplitudes of 4.9 to 6.1 mV and post-stimulus latencies of 0.89 to 3.7 ms. Immediately after surgical realization of nervis cruralis substance loss, electromyograms of muscle quadriceps were negative for all experimental animals. Electromyography showed stimulo-detection recovery in neurosphere-transplanted pigs which was partial at 180 days after lesion and almost complete by 240 days, while electromyograms of controls were still negative (Liard et al 2012). Interestingly, in neurosphere-transplanted pigs, post-stimulus latencies returned earlier to normal (i.e., at 180 days post-lesion) than electromyogram amplitude (which was fairly detectable although much lower than on intact leg).
Therefore, transplanting
4.2. Exclusively neuronal fate of grafted neurosphere cells
In order to assess the fate of transplanted neurosphere cells [53], we euthanized the experimental animals at various post-lesion intervals, sampled the experimental vein-bridged nerve segment and processed it for immunohistochemistry (fixation by 24-hour-immersion at 4°C into a buffered 4% paraformaldehyde solution, cryoprotection in 30% sucrose-containing phosphate buffer). After snap-freezing in isopentane at -45°C, serial sagittal sections were collected from each frozen vein-bridged nerve segment in a cryostat and processed for phenotypic marker immunohistofluorescence. Fluorescent dyes were chosen so as to be compared with either intrinsic green fluorescent protein (GFP) or with BrdU immunohistofluorescence, depending on the method for neurosphere cell labeling prior to transplantation [53].
At 8 days after lesion-transplantation, BrdU-positive cells were localized inside the venous tube as flattened spherical groups and were all immunoreactive for the specific marker of immature migration-prone neurons: doublecortin (DCX) [53]. At longer survival delays assessed (45 to 240 days) all neurosphere-derived cells, whether labeled with BrdU or virally-transferred GFP, expressed the specific marker of mature neurons: neuronal nuclear antigen (NeuN) or neurofilament protein NF-68 (Figure 4; modified from ref 53). By contrast, none of labeled neurosphere-derived cells coexpressed any of glial markers assessed: CNPase (for myelinating glia, i.e. oligodendrocytes or Schwann cells), S-100β and GFAP (for astrocytes), at either of post-lesional delays. Therefore
Our finding contrasts with numerous previous attempts to improve post-lesional repair of peripheral with putatively regenerating cell transplantation. The latter strategy, or cell therapy, has been attempted with primary cultures of Schwann cells [62,63], olfactory bulb enshealting cells [64], or various types of stem cells [65]. In all these studies, progenies of grafted stem cells mostly proved to be glial cells, which indirectly enhance axonal regeneration [65,66,67]. Since none of these paradigms used vascular bridge for stem cell transplantation, our results suggest that the venous wall
4.3. Graft-induced activation of intrinsic Schwann cells in the lesioned nerve
At 180 and 240 days after nerve lesion and GFP-expressing neurosphere transplantation, CNPase immunohistofluorescent labeling was much higher than in vein-bridged lesioned controls which had not received neurosphere transplantation [53]. Both the number of CNPase-immunoreactive Schwann cell processes and their labeling intensities, as quantified by computerized image analysis, were significantly higher in neurosphere-grafted nerves than in controls [26]. Our result is in keeping with a previous report showing that a venous graft favored Schwann cell proliferation after nerve lesion [75]. In our paradigm though, since controls have received a neurosphere-devoid venous graft, our results indicated that neurosphere cells emit diffusible signals that stimulate Schwann cells. Therefore,
Altogether, our novel cell therapy paradigm in adult pig promotes efficient functional recovery of a peripheral nerve after lesion with long substance loss, correlatively with genesis of new neurons in-between the lesional gap and activation of intrinsic myelinating cells.
Similar results have been obtained in adult rat by using a similar strategy (Xu et al 2012) or mesenchymal stem cells combined with acellular conduits (Zhao et al 2012, Jia et al 2012). From both our and others’ studies though (as reviewed in ref 66), a pending issue remains unaddressed: which is the long-term fate of ectopic graft-derived new neurons? The putative formation of ectopic multisynaptic neuronal nets bridging the gap between the two sides of the lesional gap, as suggested by the topographical distribution of new neurons (see above), is made plausible by previous experimental demonstrations of synaptic connectivity between ectopic stem cell-derived new neurons and host neural net, both in neurogenic [56] and non-neurogenic (Lu et al 2012) adult structures of mammalian central nervous system. However, this issue requires to be directly addressed by means of electron microscopy.
Another question arising from our results, concerns the potential long term negative effects of grafted stem cell-derived ectopic neurons. Such transplantation might indeed result into neuromas: either neoplastic neuromas deriving from the grafted proliferative stem cells (Johnson et al 2012), or non-neoplastic neuromas consisting in aberrant axonal swellings (Rajput et al 2012).
5. Future applications of these new paradigms
The results above (sections II & III) bring about a proof-of-concept for future improvements of neurological clinics. However, before it can be tested in human patients, our paradigm deserves some improvements.
5.1. Alternative source of adult neural stem cells
The direct transposition of the present cell therapy strategy to human patients would raise ethical problems, regarding either autologous sampling of patient’s subventricular zone as a source of neurogenic neurospheres, or xenotransplantation of neurospheres from adult pig SVZ due to currently discussed virological risks of porcine tissues [10]. A convenient alternative can be provided by the recently discovered neurogenic stem cells of biopsied human olfactory mucosa [84]. This tissue has been demonstrated indeed to harbor a population of mesenchymal stem cells which displays the same neurogenic properties as original neural stem cells after isolation and expansion in primary culture. To this aim, olfactory mucosa biopsies are enzymatically and mechanically separated from the olfactory neuroepithelium, and the cell suspension from the resulting tissue (olfactory chorion) is seeded in a Dulbecco’s medium (DMEM) supplemented with F12 supplement and 10% fetal bovine serum. This primary culture yields adherent proliferating cells that are self-renewable under passage into the same medium. Upon seeding into serum-free DMEM supplemented with insulin, transferrin, selenium, EGF and bFGF, olfactory chorion-derived cells grow into typical neurospheres that exhibit the same morphology, growth kinetics and multipotentiality as neurospheres from SVZ [3,85,86,87]. These
5.2. Alternative neuroguide
Another issue in our cell therapy strategy concerns the use of autologous venous shaft as a neuroguide. Although largely used in human clinics [57, 58], such procedure has indeed been reported to generate inflammatory or necrotic outcomes. This risk could be avoided by using a synthetic neuroguide which has already been shown to favor neurogenesis from transplanted neural stem cells [59,60,90,91]. The basic neuroguide structure can be manufactured by diverse methods: spinning mandrel technology, sheet rolling, injection-molding, freeze-drying, and electro-spinning.
It will be interesting to study this cell enhancement with FDA- and CE-approved neuroguides [60, 90]:
Nerve tubes from biodegradable and biological materials:
type 1 collagen =Integra NeuraGen ® ; Neuro-matrix and Neurolac [91,92,93] with retrospective studies [94];
Nerve tubes from biodegradable and synthetic materials :
poly-lactic-glycolic acid (PLGA) ;
poly-L-lactide-caprolactone (PLCL)
Neurolac® [97];polyvinyl alcohol hydrogel SaluBridge.
Nerve guide with internal multitubular architecture, which is required for bridging long nerve gaps. If we take this point of view, acellular nerve graft Axogen’Avance ® is promissing and obtained by cryocongelation and chimical treatment from human nerves [93,98]. The graft can be handled like an autograft, held by epineurium and bridge gaps up to 50 mm.
With synthetic neurotube, three-dimensional scaffold can be obtained by electrospinning and progress with nanotechnologies. This approach could be optimized with extracellular matrix (ECM) proteins or peptides (laminin-1) and neurotrophic growth factors like basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF) or nerve growth factor (NGF) [99].
Above all maybe, the use of acellular or totally synthetic conduits for nerve gap bridging is a
6. Conclusion
Thus, transplantation of neural stem cells from adult mammalian brain inside an autologous venous graft provides an efficient repair strategy, in the pig model. Our study provides the proof-of-concept for further study in human clinics. It also provides progress in basic cell biology, since the grafted exogenous neurospheres were shown to promote nerve regeneration through 2 distinct original mechanisms: i) indirect activation of intrinsic myelinating glial cells (as recently reviewed in 66), and ii) genesis of new neurons aligned in-between the two ends of the severed nerve. The latter one is totally novel in the field of post-lesional plasticity and repair of peripheral nerves. It deserves though further analysis, by deciphering the neurotransmitter phenotype of neurosphere-derived new neurons and their ultrastructural pattern of connectivity.
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