Optimization of the transplantation time-schedule when using the co-layer

In the first experiment, the newly generated plasmid construct pCAGGS-EGFP was successfully tested in vivo and there was a clear hint on the beneficial effect of the novel pretreatment protocol concerning the yield of transplanted and surviving DA neurons. Logically, we kept the track of optimizing the transplantation time-schedule when using the co-layer method.

M. Timmer, Institute of Neuroanatomy, MHH, had previously demonstrated that the highest percentage of THir cells was found in vitro and in vivo when expanded E12 VM cells were differentiated for 4 days. After longer differentiation, THir neurons exhibited in vitro very long axons and dendrites and the subsequent DA numerical and behavioral outcome in vivo was diminished (Timmer et al., 2006). As the studies on the possibilities to non-virally increase the in vivo survival of the expanded progenitors in our lab started, the differentiation interval of 4 days was integrated in the cell culture protocol of the VM NPCs after transfection (the mono-layer method) (Cesnulevicius et al., 2006). Notably, A. Nobré, PhD, Institute of Neuroanatomy, MHH, made an important observation. He showed that in the cultures where

transfected E12 were treated according to the co-culture format DA neurons

Figure 13. Experimental desing of the Exp II.

Rot I and II, apomorphine- and amphetamine-induced rotations at each of the time-points, EP, embryo preparation, A, attachment medium, P, proliferation medium, D, differentiation medium. The single numbers assign the number of days the NPCs were treated with the respective medium. (A) Time-table in weeks in vivo. (B) Cell culture conditions qualitatively compared with regard to the THir phenotype

displayed a dramatic increase in axonal sprouting after a single day in vitro, if compared to transfected E12 cells cultured according to the mono-layer method (Nobre, 2009). This meant that the THir cells treated with the co-layer method required much shorter time to exhibit a mature morphology. The time the tranfected co-layers were treated in culture could be reduced when reducing the differentiation interval and when passing the co-layer cultures directly into differentiation following the reseeding on the non-transfected bottom-layer cells post transfection. Namely, it was known from the previous in vitro experiments that the transfected NPCs require treatment with attachment medium containing fetal calf serum for one day when re-seeded on pre-coated wells.

In this context, two specified questions were addressed in the next in vivo experiment: Firstly, would considerable and comparable amounts of TH expressing cells be observed after 2 weeks in vivo if no attachment medium was used after seeding the transfected cells on the non-transfected E12-cell bottom-monolayer?

Secondly, would the TH expressing cell yield be affected if the differentiation period in vitro was shorter than 4 days?

In the second in vivo experiment (Fig. 13) twelve unilaterally 6-OHDA lesioned animals were allocated in 6 experimental groups (n=2) discerned based on the differentiation-span and medium conditions the VM progenitors were subjected to post transfection with pCAGGS-EGFP plasmid. All transplanted E12 cells were cultured for three days (1A2P), followed by detachment and transfection of the top-layer cells, which were then reseeded on non-detached sister cultures. The cell suspensions were transplanted after one (1D), two (2D or 1A1D), three (3D or 1A2D), or four (1A3D) days of further in vitro treatment, depending on the experimental group. Rats were transcardially perfused 2 weeks post implantation and brains were processed for immunohistochemistry (IHC). Evaluation of the intrastriatal DA grafts was qualitative.

Figure 14. DA cells in the E12 grafts exhibiting a range of sizes in TH-immunostained coronal sections.

Dense axonal networks present within the grafts in all three transplantation groups where E12 cells had not been treated with attachment medium for additional day (D, E, and F). By contrast, DA cells seem to cohere in a much closer distance in groups 1A2D (B) and 1A3D (C).

i) Approval of abundance of additional treatment with attachment medium Design of the Exp II allowed obtaining qualitative comparison of DA pools within intrastriatal grafts in unilateral toxin-lesioned rats, when directly comparing the THir morphology in groups transplanted with cell suspensions differentiated from a single up to 3 days after transfection with or without additional treatment with attachment medium, utilizing E12 VM NPCs obtained from the same embryo preparation-session (Fig. 14). There were qualitatively detectable clear differences in overall THir neuron population and fiber outgrowth in favor of all groups (Fig. 14-16) where pretreatment with additional day with attachment medium was abandoned. This resulted in not only comparable amounts of TH expressing cells; moreover, dense axonal networks were presented in the grafts (Fig. 15).

Thus, the co-layer cultures could be passed directly into differentiation phase post nucleofection of the top-layer sister cultures in the following experiments and the in vitro time-span prior to engraftment could be shortened.

ii) Validation of the optimal differentiation period in vitro with the novel protocol before transplantation procedure

Results in the Exp II were evaluated qualitatively with respect to the DA neuron morphology and distinctive patterns of THir neuronal outgrowths within the transplants, as well as the extent of THir processes running along to reinnervate the host striatum. The three groups, where co-layer cultures had been directly treated with the differentiation medium post nucleofection, were compared. The differentiation interval had been lasting for one (1D), two (2D) or three (3D) days.

The 2D pretreatment proved to elicit more pronounced THir connectivity within the graft, as well as graft-derived THir fiber extensions into the denervated striatum (Fig.

17). Thus the 2D pretreatment was chosen for the further co-layer experiments.

4.2 Detection and evaluation of transgene delivery 9 and 12 weeks after