Complementation of dopaminergic signaling by Pitx3–GDNF synergy induces dopamine secretion by multipotent Ntera2 cells

Abstract

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over‐secreting glial cell‐derived neurotrophic factor (GDNF). Immunocytochemistry, reverse‐transcription and real‐time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High‐performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6‐OHDA‐leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3–GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Functional dopaminergic neurons derived from human chorionic mesenchymal stem cells ameliorate striatal atrophy and improve behavioral deficits in Parkinsonian rat model

“…In general, there are several potential cell types, including human teratocarcinoma cell line Ntera2, neural stem cells, iPS cells, ES cells, MSCs, olfactory ecto‐MSCs (OE‐MSCs), and somatic cells, which could directly convert into DA neuron‐like cells (Alizadeh et al, ; Alizadeh et al, ; Alizadeh et al, ; Björklund et al, ; Choi et al, ; Eskandarian Boroujeni, Aliaghaei, Maghsoudi, & Gardaneh, ; Lindvall et al, ; Roy et al, ). For instance, Alizadeh, Kamrava, et al () used a new source of MSCs, human OE‐MSCs derived from the adult human olfactory mucosa, and differentiated these cells into DA‐neuron cells successfully by factors such as bFGF, SHH, FGF8, GDNF, and BDNF in 12 days.…”

Mechanisms of NURR1 Regulation: Consequences for Its Biological Activity and Involvement in Pathology

“…Another well-known and essential partner of the NURR1 is the homeobox transcription factor PITX3 (Table 1) [73,82]. NURR1 alone cannot drive the DAergic phenotype in meso-diencephalic DAergic neurons, due to co-repression by PSF (SFPQ; PTB-associated splicing factor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), that can interact with NURR1 and occupy its promoters target genes (Figure 2).…”

“…Hence, PITX3 is essential for the transcriptional activity of NURR1, activating its function through its response elements and allowing the development of the DAergic phenotype. This mechanism provides new insights underlying the development of midbrain DAergic neurons and stem cell reprogramming as future therapies for PD [82]. p57Kip2 (cyclin-dependent kinase inhibitor 1C (CDKN1C)) is another protein th makes heterodimers with NURR1 and is essential for normal DAergic neuron develo ment (Table 1) [74].…”

The Crucial Roles of Pitx3 in Midbrain Dopaminergic Neuron Development and Parkinson’s Disease-Associated Neurodegeneration

“…NT2derived neuron-like cells can give rise to the growth of axons and dendrites [98]. Thus, NT2 cells have long been thought of as a replacement source of mDA neurons for PD cell treatment [99]. Recent studies showed that after overexpressing Pitx3 and being exposed to GDNF, NT2 cells could release DA, indicating that Pitx3-GDNF interactions in DA signaling may promote the dopaminergic neuronal properties of NT2 cells, making it clinically applicable for cell replacement therapy in PD [99].…”