González-Gil Alfredo, Rojo Concepción, Ramírez Esther, Martín Ricardo, Suárez-Pinilla Alberto Samuel, Ovalle Susana, Ramos-Ruiz Ricardo, Picazo Rosa Ana
Department of Physiology, School of Veterinary Medicine, Complutense University of Madrid, 28040 Madrid, Spain.
Department of Anatomy and Embryology, School of Veterinary Medicine, University Complutense of Madrid, 28040 Madrid, Spain.
Biomedicines. 2025 Jun 26;13(7):1560. doi: 10.3390/biomedicines13071560.
Exploring the neurogenic potential of extraneural stem cells under the actions of proneurogenic biomolecules may enhance the success of autologous cell therapy for neurodegenerative diseases such as Parkinson's. Neural stem and progenitor cells (NSPCs) from extraneural tissues have emerged as potential sources of functional dopaminergic (DA) neurons. : This study aimed to generate DA neurons from ovarian cortical cells (OCC)-derived NSPCs to elucidate whether follicle-stimulating hormone (FSH) can enhance this process and to evaluate the electrophysiological functionality of differentiated neural cells using the patch-clamp technique. : OCC-NSPCs were differentiated towards the DA pathway during the neurosphere (NS) assay after two culture periods for cell expansion (CEP-1, CEP-2) with one of these media: M1 (positive control with epidermal growth factor, EGF, and fibroblast growth factor2, FGF2), M2 (control), and M3 (M2 with FSH, 50 ng/mL). Image analysis, morphometric evaluation, cell proliferation assays, and gene expression analysis of NSPC-specific transcripts were performed. After CEP-2, NS cells were cultured for 30 days in a serum-free medium containing Sonic-Hedgehog, FGF2, FGF8, and brain-derived neurotrophic factor (BDNF) for differentiation. At the end of culture, expression, and immunolocalization of GFAP, Olig2, NeuN, and tyrosine hydroxylase (TH) were analyzed in cells, along with patch-clamp recordings in differentiated neurons. : Cell proliferation and NS development were larger in OCC-NSPCs from groups M1 and M3 than in M2. Expression of NSPC-related transcripts was higher in M2; however, M1 and M3 cultures showed greater expression of differentiation markers , , , and . NeuN, GFAP, and TH were immunolocalized in differentiated cells and NS that were generated during differentiation. TH was localized in neural precursor cells, some neurons, core cells of small-, medium-, and large-sized NS, and in cells close to the outer cell layer of large NS, with greatest immunolocalization percentages in NS primed with FSH during CEP-1/2 (M3). Electrophysiological recordings revealed a major incidence of plateau potentials and a significant proportion of complete action potentials, reflecting successful functional neuronal differentiation. : DA precursors and functional neurons can be successfully obtained after OCC-NSPCs-directed differentiation. FSH priming during the expansion period enhances the neurogenic potential of these cells towards the DA pathway. Future research will explore the eventual therapeutic use of these findings for neurodegenerative diseases.
探索神经源性生物分子作用下神经外干细胞的神经源性潜能，可能会提高帕金森氏症等神经退行性疾病自体细胞治疗的成功率。来自神经外组织的神经干细胞和祖细胞（NSPCs）已成为功能性多巴胺能（DA）神经元的潜在来源。本研究旨在从卵巢皮质细胞（OCC）衍生的NSPCs中生成DA神经元，以阐明促卵泡激素（FSH）是否能增强这一过程，并使用膜片钳技术评估分化神经细胞的电生理功能。在神经球（NS）试验期间，将OCC-NSPCs在两种用于细胞扩增（CEP-1、CEP-2）的培养期后，用以下培养基之一向DA途径分化：M1（含表皮生长因子、EGF和成纤维细胞生长因子2、FGF2的阳性对照）、M2（对照）和M3（含50 ng/mL FSH的M2）。进行了图像分析、形态计量评估、细胞增殖测定和NSPC特异性转录本的基因表达分析。在CEP-2之后，将NS细胞在含有音猬因子、FGF2、FGF8和脑源性神经营养因子（BDNF）的无血清培养基中培养30天进行分化。在培养结束时，分析细胞中胶质纤维酸性蛋白（GFAP）、少突胶质细胞转录因子2（Olig2）、神经元核抗原（NeuN）和酪氨酸羟化酶（TH）的表达及免疫定位，同时对分化神经元进行膜片钳记录。M1组和M3组的OCC-NSPCs的细胞增殖和NS发育大于M2组。M2组中NSPC相关转录本的表达较高；然而，M1组和M3组培养物中分化标志物、、和的表达更高。NeuN、GFAP和TH在分化过程中产生的分化细胞和NS中进行了免疫定位。TH定位于神经前体细胞、一些神经元、小、中、大型NS的核心细胞以及靠近大型NS外细胞层的细胞中，在CEP-1/2期间用FSH预处理的NS中免疫定位百分比最高（M3）。电生理记录显示平台电位的发生率较高，且有相当比例的完整动作电位，反映了成功的功能性神经元分化。经OCC-NSPCs定向分化后可成功获得DA前体和功能性神经元。扩增期用FSH预处理可增强这些细胞向DA途径的神经源性潜能。未来的研究将探索这些发现最终用于神经退行性疾病治疗的可能性。
