Synergistic Effects of Combined Nurr1 Overexpression and Natural Inducers on the More Efficient Production of Dopaminergic Neuron-Like Cells From Stem Cells

Abstract

The development of dopaminergic (DA) neurons is a very complex process, and a combination of extrinsic and intrinsic factors involves their differentiation. Transcription factor, Nurr1 plays an essential role in the differentiation and maintenance of midbrain DA neurons. Nurr1-based therapies may restore DA function in Parkinson's disease (PD) by replacing damaged cells with differentiated cells derived from stem cells. Providing tissue-specific microenvironments such as brain extract can effectively induce dopaminergic gene expression in stem cells. The present study aimed to investigate the combined effects of Nurr1 gene overexpression and a neonatal rat brain extract (NRBE) induction on dopaminergic differentiation of P19 stem cells. In order to neural differentiation induction, stably Nurr1-transfected cells were treated with 100 μg/ml of NRBE. The differentiation potential of the cells was then evaluated during a period of 1–3 weeks via various methods. The initial evaluation of the cells by direct observation under a light microscope and cresyl violet specific staining, confirmed neuron-like morphology in the differentiated cells. In addition, different molecular and cellular techniques, including real-time PCR, immunofluorescence, and flow cytometry, demonstrated that the treated cells expressed pan-neuronal and dopaminergic markers. In all experimental groups, neuronal phenotype with dopaminergic neuron-like cells characteristics mainly appeared in the second week of the differentiation protocol. Overall, the results of the present study revealed for the first time the synergistic effects of Nurr1 gene overexpression and possible soluble factors that existed in NRBE on the differentiation of P19 stem cells into dopaminergic neuron-like cells.

Single-Cell RNA-Seq Reveals the Cellular Diversity and Developmental Characteristics of the Retinas of an Infant and a Young Child

“…Meanwhile, GADD45B is considered as a myeloid differentiation primary response gene ( Abdollahi et al, 1991 ). NR4A2 , also known as nuclear receptor NURR1 , plays a critical role in the differentiation and maintenance of dopaminergic neurons ( Beiki et al, 2021 ). Activator Protein-1 family member JUND is involved in regulating the transcriptional expression of genes related to cell differentiation ( Shaulian et al, 2000 ).…”

Possible role of lncRNAs in amelioration symptoms in an animal model of Parkinson’s disease following transplantation of dopaminergic cells derived from stem cells

“…However, it is possible that the factors expressed by the differentiated P19 cells markedly improve the neurodegenerative effects of toxins in PD models. It has been previously shown that when exposed to various inducers, P19 cells were able to express different factors including nestin, SYP, TH, Onecut (one cut homeobox 1), and neurotrophic factors (NTF) such as BDNF (brain-derived neurotrophic factor), NGF (nerve growth factor), and GDNF (glial cell line-derived neurotrophic factor) [14,15]. Furthermore, these cells can produce and release a highly speci c marker of DA cells, TH, in a differentiated state (unpublished data).…”

“…Wistar rat strain was obtained from Isfahan University of Medical Sciences (Isfahan, Iran) and maintained under standard situations (20 ± 2 • C, with a regular dark/light cycle and ad libitum availability in diet and drinking water). First, 28 male rats (220-280 g) were assigned randomly into four groups (Table 1): (1) Sal (control: the rats injected with isotonic saline solution); (2) Par (Parkinson's rat model: 6-OHDA-lesioned animals); (3) Par-E (Parkinson's rats under cell therapy: A line of embryonal carcinoma stem cells, P19 was transfected with pCMV3-GFPSpark vector and injected into the animals); (4) Par-N (Parkinson's rats under cell therapy: P19 ECCs were transfected with pCMV3-C-NR4A2-GFPSpark vector, containing Nurr1 gene, and injected into the animals) [15]. 2.2.…”

“…After preparing the tissue sections as explained above, they were immersed in cresyl violet solution (0.25% cresyl violet + 0.8% glacial acetic acid + 0.6 mM sodium acetate) [15]. Thereafter, the sections were rinsed, dehydrated, cleared, mounted for observation, and photographed by an optical microscope.…”

“…These cells express the pluripotency factors, which disappear after their differentiation [8,11]. Induction of neural/dopaminergic differentiation was generally applied on the P19 cell line by using natural/synthetic factors including neonatal rat brain extract (NRBE) or RA or a combination of broblast growth factor 8 (FGF8) and sonic hedgehog (Shh) [12][13][14][15]. Until the late 1970s, it was widely thought that central nervous system (CNS) treatment would never, ever be possible [16].…”