Following transplantation of foetal primary dopamine (DA)-rich tissue for neurorestaurative treatment of Parkinson's disease (PD), only 5-10% of the functionally relevant DAergic cells survive both in experimental models and in clinical studies. The current work tested how a two-step grafting protocol could have a positive impact on graft survival. DAergic tissue is divided in two portions and grafted in two separate sessions into the same target area within a defined time interval. We hypothesized that the first graft creates a "DAergic" microenvironment or "nest" similar to the perinatal substantia nigra that stimulates and protects the second graft.6-OHDA-lesioned rats were sequentially transplanted with wild-type (GFP. -, first graft) and transgenic (GFP. +, second graft) DAergic cells in time interims of 2, 5 or 9. days. Each group was further divided into two sub-groups receiving either 200. k (low cell number groups: 2dL, 5dL, 9dL) or 400. k cells (high cell number groups: 2dH, 5dH, 9dH) as first graft. During the second transplantation, all groups received the same amount of 200. k GFP. + cells. Controls received either low or high cell numbers in one single session (standard protocol). Drug-induced rotations, at 2 and 6. weeks after grafting, showed significant improvement compared to the baseline lesion levels without significant differences between the groups. Rats were sacrificed 8. weeks after transplantation for post-mortem histological assessment.Both two-step groups with the time interval of 2. days (2dL and 2dH) showed a significantly higher survival of DAergic cells compared to their respective standard control group (2dL, +. 137%; 2dH, +. 47%). Interposing longer intervals of 5 or 9. days resulted in the loss of statistical significance, neutralising the beneficial two-step grafting effect. Furthermore, the transplants in the 2dL and 2dH groups had higher graft volume and DA-fibre-density values compared to all other two-step groups. They also showed intense growth of GFP. + vessels - completely absent in control grafts - in regions where the two grafts overlap, indicating second-graft derived angiogenesis.In summary, the study shows that two-step grafting with a 2. days time interval significantly increases DAergic cell survival compared to the standard protocol. Furthermore, our results demonstrate, for the first time, a donor-derived neoangiogenesis, leading to a new understanding of graft survival and development in the field of cell-replacement therapies for neurodegenerative diseases. © 2014 Elsevier Inc.