Delayed Heterochronic Transplantation following Focal Cortical Lesion Improves Outcome

Over the past decades, many different strategies have been proposed to overcome the limited capacity of the mammalian brain to repair itself. These strategies range from cell transplantation to the administration of growth factors. These different approaches have achieved varying degrees of success, with some of them holding strong promise for translation into the clinic.

Fetal cell/tissue transplantation has been arduously studied as a potential way to repair the injured brain. Embryonic cortical neurons transplanted into the injured adult cortex were shown to differentiate into pyramidal glutamatergic neurons and form synaptic structures. Moreover, transplanted neurons survived for a long time and integrated into the host motor circuitry, extending axonal projections to different regions of the brain and even to the spinal cord with great specificity (A. Gaillard et al., 2007; Grealish et al., 2015; Michelsen et al., 2015; Falkner et al., 2016). Graft transplantation of embryonic cortical neurons may thus hold therapeutic potential and warrants further detailed analysis of its translational value.

Determination of the optimal therapeutic window for transplantation is of paramount importance for allowing effective translation to the clinic, in part because a delay between injury and transplantation is inevitable in a clinical setting. Moreover, there is growing evidence that the acute inflammatory response occurring immediately after injury hampers repair (Bonestroo et al., 2013; Silver et al., 2014). This acute response is followed by a transient proneurogenic phase when neurotrophic and growth factors, which play an important role in promoting brain repair, are upregulated (Lindholm et al., 1992; Felling et al., 2006). This phase might provide an optimal therapeutic window for transplantation because it may increase functional integration of the graft into the host tissue.

In a recently published study, Péron et al. (2017) sought to identify the optimal time for transplantation by comparing the effect of immediate and delayed transplantation of E14 motor cortical neurons on graft vascularization, survival, and contribution to long-term motor outcome. Cortical neurons from the presumptive motor cortex of embryonic day 14 (E14) mouse embryos were transplanted into the injured motor cortex of 3- to 6-month-old adult mice. Before transplantation, lesion was produced in adult mice by aspiration of the motor cortex, leaving the corpus callosum intact. In a pilot study, transplanting the graft 4, 7, or 30 d after lesion induction indicated that a delay of 7 d was the optimal time for transplantation. The results thus demonstrated that delaying transplantation within a specific time window increased graft survival and integration into host brain tissue.

The improved transplantation efficiency in the delay condition could be explained by the fact that the acute inflammatory reaction that occurs after injury decreases within a few days following the insult, thus leading to an environment more receptive for grafting. Moreover, the data show that a much longer delay is less efficient, probably due to decreased expression of neurotrophic and growth factors at the lesion site (Nieto-Sampedro et al., 1983, 1984). Together, their data suggest the existence of a temporal window after injury induction, when there is an optimal balance between inflammation and prorepair factors, which improve transplantation outcome. It would be valuable for future studies to investigate the molecular mechanisms underlying the enhanced graft survival rate after delayed transplantation.

Having determined in their pilot study that 7 d delay was the optimal time for transplantation, Péron et al. (2017) performed additional studies to compare the effect of 7 d delay and immediate transplantation on survival and integration of grafted cells into host brain tissue at 4, 7, and 14 d after transplantation. Their results showed that delaying transplantation increased cell proliferation, without increasing apoptosis, consequently leading to increased graft size in the delayed condition. Delayed graft transplantation also led to an increase in donor-derived blood vessels, resulting in a transient improvement of graft vascularization at 4 d after transplantation. In vivo imaging using two-photon microscopy further confirmed that vascularization in the delayed condition consisted of both regenerated host vessels and donor blood vessels. The host vessels were more swollen 7 d after transplantation in the no-delay group than in the delayed transplantation group, suggesting that ongoing inflammation was present in the no-delay group. Péron et al. (2017) conclude that delaying transplantation allowed endothelial progenitor cells present within the graft to survive and differentiate to form vessels. The imaging protocol the authors established to assess vasculogenesis in vivo should be used in the future to study vascularization after transplantation in more detail (e.g., to determine whether donor and host blood vessels connect).

Delaying transplantation for 7 d after injury also improved integration of the transplanted neurons. Neurons transplanted at this stage not only extended fibers to the ipsilateral caudate-putamen and cortex, as in the no-delay condition, but also through the corpus callosum to the contralateral side and even to spinal cord by 90 d after transplantation. This finding further demonstrates that 1 week delay in transplantation improves outcome. It further supports the idea that there is an optimal time window during which the balance between proneurogenic and proinflammatory cues tilts toward repair. In this regard, the improvement in axon growth could be partially due to decreased inflammatory signals surrounding the lesion cavity, which, if present, would form an inhibitory barrier for axon extension.

The observation of regeneration of axonal projections in the adult brain suggests that axon guidance cues are reexpressed after injury. Indeed, chemorepellent axon guidance cues, such as Sema3a in the brain (Pasterkamp et al., 1999) and Wnts in the spinal cord (Onishi et al., 2014), were found to be increased after injury. Alternatively, it has been shown in the spinal cord, that after Wallerian degeneration, axons regenerate by following the path left behind by the degenerated axon (Wang et al., 2012). One could thus postulate that a delay following injury induction allows for complete axon degeneration and debris clearance, and may thus facilitate axon regrowth along the vacated tract.

Péron et al. (2017) used in vivo intracortical microstimulation to assess long-term functional integration of grafted cells with host motor circuitry. Directly assessing the functioning of grafted cells is important because having a behavioral test as the sole read-out for functional integration of grafted neurons could be misleading. Indeed, improved motor outcome has been shown to occur after stroke as a result of remodeling of existing intact cortical connections, rather than via the integration of newborn neurons into the local network (Dancause et al., 2005). By electrically stimulating the graft, the authors showed that the grafted region was indeed able to evoke motor responses in the affected limbs, thereby confirming the functional integration of grafted neurons into preexisting circuitry.

The finding that host blood vessels grow inside the graft, highlights an interesting issue, namely, whether host and graft cells intermingle (i.e., do grafted endothelial cells or neurons invade surrounding host brain tissue or do grafted cells remain segregated from the surrounding environment). It has been shown that some transplanted embryonic neurons migrate away from the graft and invade surrounding brain tissue (F. Gaillard et al., 2000). It would be interesting to assess whether these grafted neurons found outside the graft are also able to generate efferent and afferent connections within the host circuitry. The in vivo 2-photon microscopy technique would allow studying this graft–host interaction in more detail. This approach would help to determine whether endogenous neural progenitor cells contribute to increased graft size and whether grafted glutamatergic projection neurons form appropriate intracortical connections with host glutamatergic neurons and receive thalamic input. In this context, it would be valuable to determine whether delaying transplantation also improved cortical layer formation within the graft, which is an important read-out to assess proper integration of newborn neurons.

The observation of host blood vessels growing into the graft is of interest to both the regenerative and developmental field and underlines the importance of further dissecting host–graft assimilation. The combination of in vivo 2-photon microscopy and intracortical microstimulation used by Péron et al. (2017) provides the foundation for future studies to dissect the mechanisms underlying increased graft integration and the presumptive symbiosis between graft and host. Their demonstration of a long therapeutic window and long-term functional improvement brings graft transplantation a step closer to clinical translation once an adequate cell/tissue source has been established.