Cell Replacement Therapy for Parkinson's Disease

How Close Are We to the Clinic?

Javier Ganz; Nirit Lev; Eldad Melamed; Daniel Offen


Expert Rev Neurother. 2011;11(9):1325-1339. 

In This Article

Dopaminergic Neuron Induction Mechanisms

The success of emerging CRT for PD will depend on other issues, such as the implantation site, environment modification or patient selection, the accurate combination of chosen stem cells type and the methodology or differentiation protocol that will be applied. Within a decade, neuroscientists have gathered a wealth of information about the midbrain DA system and have identified many molecular processes and mechanisms that underlie midbrain DA neuron development, maintenance and function. To date, several protocols have been designed to induce the desired A9 DA neuron phenotype using the abovementioned cells (Table 1). These protocols intend to simulate the natural development process induced by intrinsic and extrinsic factors involved in DA neurogenesis. Within the extrinsic factors, the most widely used are soluble proteins, such as SHH, FGF-2 and -8 and members of the Wnt family. SHH is a morphogenetic factor secreted from the floor plate with a crucial effect on the ventral midbrain during development, and it induces the proliferation of DA neuron precursors.[97,98] In the same way, FGF-8, produced by the isthmus organizer, also plays an important role in DA neuron specification and promotes DA neurogenesis.[99] Members of the Wnt family are additional factors produced and secreted in the midbrain development, which promote activities such as precursor proliferation and neural differentiation.[100]Wnt signaling through the action of Wnt-1, -3a and -5a has been reported to be needed for the establishment of the midbrain–hindbrain regions and are involved in activating engrailed (EN) genes, which are necessary for later stages of midbrain DA neuronal development. Moreover, mutant Wnt mice showed a loss of most midbrain DA neurons and ectopic expression of Wnt-1 and -5a in NSCs lead to DA differentiation and NURR1 positive cells.[101–103] Other growth factors, such as BDNF, GDNF, EGF and TGF-β, have been used and have shown a DA neuron inducer activity.[90] Chemical inducers are also considered to be extrinsic factors that enhance DA neuron formation. Among these factors, RA, dbcAMP, IBMX, AA and BHA are currently the most effective inducers used.[39,90,104] To certain stem cell sources, extrinsic induction can be sufficient to generate DA neurons, but in cases of stem cells that are not naturally prone to become midbrain DA neurons, this process seems to be more difficult, inefficient and incomplete. Induction by intrinsic factors has facilitated and improved DA neuron generation. Genetic manipulation, mainly by lentiviral transduction, has demonstrated that ectopic insertion of transcription factors represented by homeodomain proteins, proneural genes and genes involved in epigenetic control, effectively induce a DA neuron phenotype.[68] The development of mice deficient in PITX3, LMX1A/B, EN1, EN2, neurogenin 2 (NGN2) and the orphan nuclear receptor 1 (NURR1) has facilitated the development of a map that specifies gene–function relationships during midbrain DA neurons differentiation.[105] The expression of the gene coding for LIM homeobox transcription factor 1 (LMX1A), has been reported to be both necessary and sufficient for the induction of the midbrain DA phenotype in midbrain neuroepithelial cells, ESCs and MSCs, an effect that can be enhanced by extrinsic factors.[86,106] Moreover, the neurotransmitter phenotype is partly determined by the transcription factor NURR1, which regulates several proteins that are required for dopamine synthesis and regulation, such as TH, vesicular monoamine transporter 2 (VMAT2), dopamine transporter (DAT) and RET receptor tyrosine kinase (cRET).[107,108] Overexpression of PITX3 and FOXA2, other transcription factors involved in DA specification, was seen to actively assist NURR1 and LMX1A to induce human ESC and NSC terminal maturation to midbrain DA neurons.[109–111] Currently, most protocols rely on early induction through SHH, FGF-8, WNT-1/-5A, TGF-β and RA, which are often combined with the introduction of transcription factors, such as LMX1A and NURR1. The generated cells should be monitored for the expression of NURR1, EN1, EN2 and the midbrain DA neuron-specific gene PITX3, which display the full DA phenotype. In order to achieve rapid and safe clinical translation, transgenesis must be preferably avoided until safer gene delivery methods can be developed.