We show that human NPC transplantation at 1 week after stroke significantly improved functional recovery in several behaviour tests and that this recovery correlated with human NPC-enhanced changes in dendritic and axonal structural plasticity. Human NPCs not only elicited these plasticity changes in the injured hemisphere but also helped recruit the uninjured hemisphere. Moreover, we demonstrate for the first time that human NPC transplantation enhanced recovery of stroke-impaired axonal transport. This is significant given the vital role of axonal transport for neuron function, survival and plasticity. Using co-culture assays, we could mimic these human NPC-induced effects on plasticity and transport in vitro, and through neutralization studies we identified four secreted factors that mediate these human NPC-induced effects. These data suggest that transplanted human NPCs enhance post-stroke recovery by secretion of factors that enhance endogenous brain repair mechanisms induced after stroke injury.
Human neural precursor cells enhance the innate repair capacity of the brain
There is compelling evidence from both patient and animal data that the brain undergoes reorganization and rewiring of surviving circuits after ischaemia and this is postulated to underlie the spontaneous recovery observed after stroke (Dancause, 2006
; Murphy and Corbett, 2009
; Benowitz and Carmichael, 2010
). Such restorative neuronal plasticity changes include an increase in afferent and efferent connections in both ipsi- and contralesional brain regions, resulting in part from changes in dendritic and axonal sprouting of surviving neurons.
Chronic changes in dendritic structural plasticity after stroke have been reported with increased contralesional layer V dendritic branching peaking at 18 days post-stroke (Jones and Schallert, 1992
), while ipsilesional layer III branching was decreased (compared with uninjured animals) at 9 weeks post-stroke (Gonzalez and Kolb, 2003
). Our data show for the first time that at 3 weeks post-stroke, human NPCs enhance dendritic length and arborization in layer V cortical neurons in both the ipsi- and contralesional cortex (despite the latter being remote from the site of transplantation), and this coincided with the onset of human NPC-induced functional recovery. Enhanced contralesional dendritic plasticity after stroke is thought to be due to compensatory increased use of the unimpaired limb (Jones and Schallert, 1994
). However, this is not apparent in the human NPC-treated animals; despite enhanced contralesional plasticity they exhibit less relative use of the unimpaired limb than vehicle controls. Thus, the significance of human NPC-enhanced contralesional dendritic plasticity and its importance for early recovery remains to be determined. Of note, vehicle injection was detrimental to dendritic plasticity at 2 weeks post-injection with recovery by 4 weeks. The human NPCs appeared to prevent this injection injury, suggesting neuroprotective activity, be it direct or indirect. This raises the question whether this neuroprotective effect is a significant component of human NPC-induced recovery in the first few weeks following transplantation.
Human NPC-induced dendritic changes in the contralesional cortex were transient returning to baseline by 5 weeks post-stroke, thus indicating that human NPCs could not prevent the contralesional branch regression previously reported (Jones and Schallert, 1992
), and that contralesional dendritic changes are not necessary for continued recovery at later time points. In contrast, ipsilesional changes (i.e. in the human NPC-grafted hemisphere) were maintained at 1 month post-transplantation with no sign of abating, suggesting that local effects of the human NPCs are necessary to maintain dendritic changes. The biggest change in dendritic structural plasticity was observed in basilar dendrites where the majority of synaptic inputs are found (Larkman, 1991
); however, the significance of this for neuron function remains to be elucidated (Nevian et al., 2007
; Spruston, 2008
). The pattern of early dendritic changes in the contralesional cortex followed by a switch to more dominant changes in the ipsilesional cortex at later times is reminiscent of brain remapping results in patients and animals. These remapping studies show that stimulation of the injured limb early after stroke recruits the contralesional cortex and this switches back to the ipsilesional cortex at later time points (Dancause, 2006
; Benowitz and Carmichael, 2010
). It would thus be of interest to determine the significance of these dendritic changes to such remapping data.
Axonal sprouting occurs after stroke with new projections thought to target areas denervated by the stroke injury (Benowitz and Carmichael, 2010
). In rodent and primate models of ischaemic cortical injury, such sprouting has been observed locally around the infarct area (Carmichael et al., 2001
; Conner et al., 2005
; Dancause, 2006
; Li et al., 2010
), and interhemispheric axonal outgrowth from the intact cortex to the injured hemisphere has also been observed (Carmichael, 2008
). Our BDA axonal tracer data suggest that human NPC transplantation promotes this interhemispheric cortical sprouting in corticocortical, corticostriatal and corticothalamic pathways. These data corroborate previous reports using human umbilical cord blood cells (Xiao et al., 2005
) and human embryonic-derived neural progenitor cells (Daadi et al., 2010
) in immunosuppressed or immunocompetent animals, suggesting that this phenomenon is independent of stem cell type used and T cell status of the host. Consistent with enhanced axonal sprouting, human NPCs also enhanced expression of the axonal growth cone protein GAP-43 in the same regions. However, GAP-43 is not purely a marker of regenerating axons as it is also expressed on non-neuronal cells such as astrocytes and oligodendrocytes (Carmichael, 2008
); the importance of this for regeneration is not understood.
Human NPC transplantation also enhanced stroke-induced remodelling of cortical–spinal tract axons originating from the contralesional cortex (i.e. intact corticospinal tract). The fact that the number of BDA-labelled fibres not only increased in the ipsilateral dorsal funiculus (intact corticospinal tract) but also in the contralateral dorsal funiculus (injured corticospinal tract) implies that human NPCs may enhance collateral (cross midline) sprouting of the intact corticospinal tract into denervated regions of the spinal cord (Chen et al., 2002
; Liu et al., 2008
). Marrow stromal cell treatment enhanced this collateral sprouting (Liu et al., 2008
), but unlike human NPC treatment, marrow stromal cells had no effect on fibre density in the intact corticospinal tract (Liu et al., 2008
). The human NPC-induced changes in both corticospinal tract and transcallosal axonal sprouting statistically correlated with human NPC-enhanced functional recovery; a similar correlation was found between recovery and marrow stromal cell-induced corticospinal tract sprouting (Liu et al., 2008
). Together, these data imply that human NPC-induced axonal plasticity is an important mechanism for human NPC-induced recovery, although a direct causal link remains to be demonstrated.
A key finding of our study is the ability of human NPCs to enhance recovery of axonal transport after stroke. This has major implications for recovery of white matter injury. Axonal transport is disrupted after ischaemia (Kataoka et al., 1989
; Valeriani et al., 2000
; Wakita et al., 2002
) and is indicative of axonal damage. Amyloid precursor protein is commonly used as a marker of impaired transport and axonal degeneration (Stone et al., 2000
; Valeriani et al., 2000
) as it is constitutively expressed in neurons and normally subject to fast axonal transport (Koo et al., 1990
). However, under pathological conditions, amyloid precursor protein accumulates in axons, which can be both a consequence and a cause of disrupted transport (Stone et al., 2000
; Gunawardena and Goldstein, 2001
; Salehi et al., 2006
). Our results confirm that stroke induces amyloid precursor protein accumulation in the corpus callosum and it remains elevated at 6 weeks post-stroke, implying extended perturbation of axonal transport. Human NPC transplantation reduced amyloid precursor protein accumulation and accelerated the decrease of amyloid precursor protein over time, which strongly suggests that human NPCs enhance recovery of fast axonal transport after stroke. Furthermore, by direct measurement of vesicle transport in cultured cortical neurons, we confirmed that human NPCs can enhance axonal transport. This is a significant finding as axonal transport is fundamental to neuron function, not only for proper functioning and survival of existing axons but also for plasticity changes such as axonal sprouting and synaptogenesis. Therefore, human NPC-induced restoration of impaired axonal transport after stroke may not only enhance the function of existing fibre tracts but may also be a key upstream event of human NPC-induced structural plasticity.
Given that axonal tracers-like BDA undergo fast axonal transport, this questions the interpretation of tract tracing data; is increased axonal labelling really indicative of a greater number of axons or does it merely reflect increased axonal transport? Because axonal sprouting, transport and survival are somewhat co-dependent, it probably represents a combination of events. Consistent with this, human NPCs enhanced both axonal transport and neurite outgrowth (measured directly) in vitro. Regardless of mechanism, increased BDA labelling probably signifies enhanced fibre tract integrity in vivo whether this is due to increased numbers of axons or better functioning (transport) of axons. The reduced atrophy of the corpus callosum observed in human NPC-treated animals provides additional indirect evidence for improved fibre tract integrity.
Identification of putative secreted factors involved in human neural precursor cell-mediated plasticity
The majority of the human NPCs remained in an immature state at 5 weeks post-stroke, therefore, differentiation into functionally mature cells is not necessary for human NPC-induced recovery. Instead, it is postulated that transplanted human NPCs elicit their effects through secretion of relevant factors. Using in vitro
co-culture systems in which human NPCs mimicked the effects observed in vivo
, we identified secreted molecules putatively involved in human NPC-induced axonal transport and neurite sprouting. We found that thrombospondins 1 and 2, VEGF, Slit, but not SPARC, were important for human NPC-induced dendritic and axonal outgrowth. SPARC may require other cells (e.g. oligodendrocytes) to potentiate its effects on neurite growth (Au et al., 2007
). Human NPCs also increased neuronal survival; whether this is related to their effects on neurite outgrowth is unclear. However, several mechanisms may be involved as depletion of thrombospondin 2 and Slit only affected neurite outgrowth without affecting cell survival. Only VEGF was important for human NPC-enhanced axonal transport. VEGF has previously been reported to be transported by axons (Storkebaum et al., 2005
), whether this is important for its effects on axonal transport remains to be determined.
Because most antibodies used in these experiments may bind both human and rodent forms of the protein, further studies are required to determine whether direct secretion of these factors by the human NPCs or human NPC-induced secretion of these factors by the primary rat cultures is important for the observed effects. However, neutralization of VEGF was achieved using Avastin, which binds human but not rodent VEGF (Ferrara et al., 2004
), strongly implying that direct secretion of VEGF by human NPCs plays a significant role. Furthermore, using a small interfering RNA knockdown approach, Yu et al. (2008
) reported that thrombospondin 1 was important for marrow stromal cell-induced neurite outgrowth in retinal ganglion cell cultures, thus strengthening the idea that direct secretion of thrombospondin by human NPCs is involved.
Whether these factors are also important for the in vivo
effects of human NPCs remains to be determined but at least many of them are expressed by the human NPCs in the stroke brain. We have previously reported the importance of stem cell-secreted VEGF for human NPC-induced functional recovery (Horie et al.
, 2011). Thrombospondins 1 and 2 are also strong candidates since growing evidence suggests the importance of thrombospondins after stroke as they are upregulated after cerebral ischaemia (Lin et al., 2003
), and we recently reported that thrombospondins 1 and 2 knockout mice have reduced functional recovery and axonal sprouting after stroke (Liauw et al., 2008
). Thus, these human NPC-secreted factors warrant further investigation, but clearly other modulatory factors will also be involved (Carmichael, 2008
Human NPCs have clinical potential for stroke therapy and we (Svendsen) have a clinical grade bank of foetal-derived human NPCs, which makes this a realistic goal. Understanding the mode of action of human NPCs in the post-ischaemic brain will be important for the successful translation of cell transplantation strategies to the clinic. For example, if modulation of host brain plasticity is a major human NPC mechanism of action, this could dictate the best time to transplant cells after stroke; ‘network relearning’ occurs within weeks of stroke and continues for several months, making it a good therapeutic target with a large time window of intervention. Furthermore, knowing what changes the human NPCs elicit in the brain offers useful surrogate indicators of transplanted cell activity. For example, changes in fibre tract integrity can be monitored non-invasively with diffusion tensor MRI, which can evaluate cell therapy efficacy and also highlight potential side effects resulting from too much or inappropriate human NPC-induced plasticity. Moving from the bench to the clinic also raises cell manufacturing issues, in particular designing bioassays to predict clinical efficacy of the cell product. Our data imply that human NPC-induced changes in neurite sprouting and axonal transport would be useful predictors of cell efficacy. Therefore, the in vitro
assays we developed here to test these parameters, with their potential for high throughput, could serve as useful bioassays. Finally, the translational feasibility of human NPC therapy will depend on many variables including the difference in scale between the rodent and human brain which may affect the ability of human NPCs to induce effective plasticity. Furthermore, patients experience a rehabilitative or enriched post-stroke environment compared to laboratory animals, which per se
is reported to enhance plasticity and recovery in rodents (Biernaskie and Corbett, 2001
; Wurm et al., 2007
). Therefore, demonstrating that human NPCs can further enhance recovery in rats under enriched conditions will be critical (Mattsson et al., 1997
; Hicks et al., 2007
). These issues warrant further investigation as cell therapies move to the clinics.