BIOGRAFÍAS: Vicky:

Despite the issues of GID, early clinical trials in individuals showed the potential benefits of cell transplantation as a therapeutic approach. However, there are significant sustainability issues in using foetal cell transplantation as a main stream therapy. More than one piece of VM and therefore more than one foetus is needed to transplant into the striatum of one patient, 4-6 pieces per hemisphere is typically required. There are logistical problems to obtaining enough VM pieces with the required properties like the appropriate age, quality, and availability at the same time (Brundin et al. 2010). In addition, changes in the process of many terminations from largely surgical to medical involving the use of hormone based medications is used. The impact of this shift in procedure on the viability of VM dopaminergic neurons was uncertain. However, specific study have been carried out to confirm that products from medical terminations of pregnancy can be used (Kelly et al. 2011). Moreover, based on religious and ethical reasons, some countries have banned the use of foetal tissue while others, who have accepted their use, have introduced legislation (the EU Human Tissue

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Act) which adds significant logistical hurdles to obtaining the tissue, around consent, management and tracking of the tissue. In addition, the great demand for using foetal tissue, that would emerge from a more widely available therapy, raises concerns about another crucial moral issue, the motivation for elective abortions (Boer & Peschanski 1994).

Alternative sources of dopaminergic neurons are therefore being explored. Pluripotent and multipotent stem cells have been looked to produce dopaminergic neuros that can replace VM cells transplantation. Embryonic stem cells (ESCs) is one of the pluripotent stem cells that is effectively generates dopaminergic neurons for transplantation (Kirkeby et al. 2012)(Kriks et al. 2011). These cells are created from inner mass of the blastocyst of early stage embryos and have proliferative capacity for extended periods of expansion with the maintenance of karyotypic stability in vitro (Amit et al. 2000). These cells can theoretically therefore provide a limitless number of cells for transplantation (Xiao et al. 2006). Protocols have been established to produce the ESCs under good manufacturing practice (GMP) and for cell banking (Tabar & Studer 2014). Another type of pluripotent stem cell successfully using in the generation of dopaminergic neurons is induced pluripotent stem cells (iPSCs) (Sundberg et al. 2013; Doi et al. 2014). The iPSCs are generated from reprogramming the adult fibroblast and they have the same proliferative and self-renewal potency of ESCs (Takahashi & Yamanaka 2006; Takahashi et al. 2007). They can be used for autologous transplantation providing the advantage of minimum immunological complications, however it also has disadvantage of the intrinsic vulnerability to the patients’ main pathology. The other reprogrammed stem cells used for developing dopaminergic neurons is the induced neurons (iNs) (Kim et al. 2011). These cells directly converted from fibroblast to other differentiated cells without passing the pluripotent stage (Vierbuchen et al. 2010). Unlike pluripotent stem cells, they lack the limitless self-renewal capacity but can still produce an expandable source of neurons (Grealish et al. 2016). Foetal brain neural stem cells NSCs are a multipotent stem cell isolated from the brain of embryos and can be differentiated into neurons, astrocyte and oligodendrocyte. The isolated NSCs from the midbrain of embryos have been successfully converted to dopaminergic neurons. However, these cells need longer time of expansion in culture with less supply of cells and lower differentiation capacity (Yasuhara et al. 2006; Madhavan et al. 2012).

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In principle, dopaminergic neurons derived from stem cells can solve many of the practical and ethical challenges of primary foetal tissues, they can be prepared in the lab, scaled up to high levels of production, and provided ready for the patients at time of surgery with fewer ethical hurdles. However, stem cell derived dopaminergic neurons have yet to demonstrate their ability to compete with primary cells in terms of efficacy and that they can be safely hosted by the PD brain. As yet there have only been studies in animal models and there is some way to go to demonstrate long term survival and efficacy, before going into clinical trials.

The main requirements of the transplanted cells are to be safe and able to survive, innervate a significant area of the striatum, form appropriate synapses and function adequately to produce sufficient dopamine to elicit recovery of motor deficits. Initially problems emerged in the technology and understanding of differentiation protocols to guide the cells towards a dopaminergic phenotype and in the timing of transplantation to create viable grafts. Various protocols have been designed and improved to generate reliable populations of dopaminergic neuron that have a midbrain phenotype of in term of transcriptional profile, protein expression, electrophysiological activity, and dopamine release. Early experiments succeeded in generating dopaminergic neurons from hESCs. However, the generated dopaminergic neurons didn’t carry the essential protein expression of the midbrain dopaminergic neurons like LIM homeobox transcription factor 1, alpha (LMX1A) and forkhead box protein A2 (FOXA2) (Yang et al. 2008; Park et al. 2005) (Emborg et al. 2013). This was followed by protocols directing the fate of the embryonic stem cells to floor plate using dual SMAD inhibition followed by the induction of midbrain regional specification of dopaminergic neurons. Now, there are well described protocols to get authentic dopaminergic neurons which are indistinguishable from midbrain equivalent primary cells (Kriks et al. 2011) (Kirkeby et al. 2012). These cells can achieve behavioural recovery in different animal models of PD. For instance, Kriks and colleagues have described protocols based on directing the hESC to be floor plate derived dopaminergic neurons precursors using SHH and WNT signalling to yield dopaminergic neurons over a period of 25 days. These neurons showed a robust survival and behavioural functioning in three different animal models of PD (6-OHDA lesioned rat, 6-OHDA lesioned -mice and MPTP-treated non-human primates) (Kriks et al. 2011). Another successful protocol described by Kirkeby and colleagues is based on embryoid body formation with dual

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SMAD inhibition followed by dose-dependent activation of WNT signalling and SHH activation to generate dopaminergic neurons. Similarly, these have been shown to be capable of reversing motor deficit in the 6-OHDA lesioned rat (Kirkeby et al. 2012). In comparing the efficiency of dopaminergic neurons derived from stem cells and human primary foetal cells, both grafts have the same apparent efficacy in reversing motor deficits (Grealish, Diguet, et al. 2014). Similar success with alternative ‘starter cells’ have also been successful in generating dopaminergic neurons and producing behavioural efficacy in animal models. This includes reprogrammed cells including induced pluripotent stem cells (iPSCs), direct conversion into induced neurons iNS and induced neural progenitor cells iNPCs (reviewed in (Grealish et al. 2016). Currently there is a global consortium under the name of “G-force PD” which shares the challenges and the solutions to pave the way for clinical trials to transplanting dopaminergic neurons directed from stem cells (Barker et al. 2015). Recently a company called “International Stem Cell Corporation (ISCO)” declared that they were conducting a clinical trial in the Royal Melbourne Hospital in Melbourne, Australia in which they are transplanting neural stem cells differentiated from a pluripotent parthenogenetic cell (hpNSC) in moderate to severe PD patients (International Stem Cell Coporation, 2015). This trial has created a lot of conflict in the field, especially as there is no clear evidence from pre-clinical studies about the safety and efficiency of these cells. One concern is that these cells express PAX6, while midbrain dopaminergic neurons are negative for this particular marker, raising the issue of what phenotype of the cells transplanted and the anticipated mechanism of action of these cells (reviewed in (Barker et al. 2016). The translation of stem cells as a source to transplantation in PD to clinical trial should be considered with caution and takes all the lessons from the previous clinical trials of primary cells transplantation to avoid possible inconsistency results that would have negative impact on the future of cell therapy. Several important features of stem cell transplantation have not yet been fully explored in the basic animal models deployed thus far.