Dose‐dependent effect of mesenchymal stromal cells co‐grafted with dopaminergic neurons in a Parkinson's disease rat model

Abstract A major limiting factor for cell therapy in Parkinson's disease is the poor survival and reinnervation capacity of grafted dopaminergic neurons, independently of the cell source. Mesenchymal stromal cells (MSCs) have high capability to regulate the local environment through the release of trophic, antiapoptotic and immunomodulatory factors. In this work, we investigated whether co‐grafting of MSCs could improve the survival and reinnervation ability of dopaminergic precursors transplanted in animal models of Parkinson's disease. Rats with total unilateral dopaminergic denervation were grafted with a cell suspension of rat dopaminergic precursors (500,000 cells) with or without a high (200,000 cells) or low (25,000 cells) number of MSCs. Eight weeks after grafting, rats were tested for motor behaviour and sacrificed for histological analysis. Our results showed that the survival of dopaminergic neurons and graft‐derived striatal dopaminergic innervation was higher in rats that received co‐grafts containing a low number of MSCs than in non‐co‐grafted controls. However, the survival of dopaminergic neurons and graft‐derived dopaminergic reinnervation was lower in rats receiving co‐grafts with high number of MSCs than in non‐co‐grafted controls. In conclusion, co‐grafting with MSCs or MSCs‐derived products may constitute a useful strategy to improve dopaminergic graft survival and function. However, a tight control of MSCs density or levels of MSCs‐derived products is necessary.

significant limitations including the survival of grafted dopaminergic neurons, which is very poor independent of the source of dopaminergic neurons. 2,3 Dopaminergic cells from induced pluripotent stem cells (iPSCs) or embryonic stem cells are being investigated as alternative cell sources for PD patients. 4,5 However, strategies to improve the survival of grafted cells and improve host reinnervation must be developed.
Mesenchymal stromal cells (MSCs) are multipotent cells originally isolated from the stroma of the bone marrow. 6 Different studies have shown their capability to regulate the local environment through the release of immunomodulatory, antiapoptotic and trophic factors.
These properties make them an attractive cell source for repairing damaged tissue. 7 Consistent with this, we have previously observed that conditioned medium derived from bone marrow MSCs increases the viability of dopaminergic cells of rat and human origin in cultures. 8 However

| MATERIAL S AND ME THODS
Young adult rats were subjected to maximal unilateral dopaminergic denervation with 6-hydroxydopamine (6-OHDA). One month after 6-OHDA injections, rats with maximal lesions (dopaminergic depletion >90%) were identified in a rotometer and confirmed with the cylinder test. One week later, rats showing behavioural criteria for maximal dopaminergic denervation were selected for transplantation (ie to investigate the effect of MSCs on the survival of grafted dopaminergic precursors). In a first series of experiments, rats with maximal dopaminergic denervation were grafted with dopaminergic precursors derived from foetal ventral mesencephalon (500,000 cells; 13 days of gestation, E13; VM; n = 5 rats, control group) or co-grafted (n = 6 rats) with 500,000 cells from the same VM suspension and a high number (200,000 cells) of MSCs (VM + high MSC-group). The number of MSCs was determined on the basis of previous studies (see Appendix S1).
In a second series of experiments, rats with maximal dopaminergic denervation were grafted with 500,000 cells from a VM suspension (n = 5 rats; control group) or co-grafted (n = 5 rats) with 500,000 cells from the same VM suspension and a low number  Cells showing red fluorescence revealed the presence of MSCs in the grafts ( Figure 1C,C′). Surprisingly, the total number of TH-ir neurons was significantly lower in VM + high MSC-grafted rats than in the rats transplanted with VM tissue alone ( Figure 1F). However, the average graft volume in the VM + high MSC-grafted group was not significantly different from that observed in VM-grafted rats (0.351 ± 0.023 mm 3 and 0.454 ± 0.065 mm 3 , respectively). The density of TH-ir fibres in the reinnervation area ( Figure 1G) and the size of the reinnervation area ( Figure 1H  Rotational values (A) for both grafted groups did not differ significantly between groups. In the cylinder test (B), VM-grafted rat scores were significantly better than in the VM + high MSC-grafted group. Immunoreactivity for tyrosine hydroxylase (TH) in rats subjected to dopaminergic denervation and co-grafting is not enough to induce functional differences or, more probably, that the sensitivity of these basic behavioural tests is not sufficient to detect a difference in the behavioural improvement.

| DISCUSS ION
The lack of alternative sources to dopaminergic neuroblasts from the  MSCs may induce MSC senescence, damaged mitochondrial transfer or dysregulation of pro-inflammatory cytokine production. [18][19][20] In any case, the results reveal that further studies are necessary to know the exact proportion of dopaminergic/MSCs that leads to the highest neuroprotective effects. In addition, future studies are necessary to identify MSCs-derived products that are responsible for the positive effects and may be used as a substitute for MSCs.
However, the present results suggest that the dose must be tightly adjusted to the number of grafted dopaminergic cells.