Generation of functional dopaminergic neurons from human spermatogonial stem cells to rescue parkinsonian phenotypes

Abstract

Background Recent progress in the induced generation of dopaminergic (DA) neurons from different types of stem cells or reprogrammed somatic cells holds tremendous potential for the treatment of Parkinson’s disease (PD). However, the lack of a reliable source for cell replacement therapy remains a major limitation in the treatment of human neurological disorders. Additionally, the current protocols for in vitro differentiation or cell reprogramming to generate human DA neurons are laborious, time-consuming, and expensive, and efficient conversion of human spermatogonial stem cells (hSSCs) to functional DA neurons has not yet been achieved. Methods Primary hSSCs from testicular tissues of patients were exposed to an improved induction system, which consisted mainly of olfactory ensheathing cell conditioned culture medium (OECCM) and a set of defined cell-extrinsic factors and small molecules. Morphological changes were assessed, along with the expression of various DA neuron phenotypic markers (e.g., Tuj-1, TH, Nurr1, DAT) and several critical pro-DA neurogenesis effectors (e.g., EN-1, Pitx3, Foxa2, Lmx1a, Lmx1b, and OTX2). In addition, transcriptome analysis was used to further evaluate the genetic similarity between the artificially differentiated DA neurons and genuine ones. Concomitantly, the functional properties of converted DA neurons including synapse formation, dopamine release, electrophysiological activity, and neuron-specific Ca 2+ signaling images were determined. Finally, hSSCs in the early stage of induction were evaluated for survival, differentiation, migration, tumorigenicity in the mouse striatum, and improvement of functional deficits in MPTP-induced PD animals. Results The hSSC-derived neurons not only acquired neuronal morphological features but also expressed various phenotypic genes and protein characteristic of DA neurons and several effectors critical for pro-DA neurogenesis. Strikingly, as the period of induction was prolonged, expression of the critical molecules for DA neuron epigenetic status gradually increased while hSSC-specific markers sharply decreased. After 3 weeks of induction, the transdifferentiation efficiency reached 21%. In addition, hierarchical clustering analysis showed that the differentiated DA neurons closely resembled genuine ones. Furthermore, the hSSC-derived neurons gained sophisticated functional properties of wild-type DA neurons, and pro-induced hSSCs efficiently survived, migrated, and differentiated into DA neurons without tumorigenesis after transplantation into mouse striatum, leading to improvement of functional deficits in PD animals. Conclusions The results showed that, using the present improved straightforward approach, hSSCs could acquire DA neuron morphological features and functional properties and rescue parkinsonian phenotypes. Our strategy for the conversion of hSSCs into DA neurons is very efficient and thus may p...

Chemical and Biological Molecules Involved in Differentiation, Maturation, and Survival of Dopaminergic Neurons in Health and Parkinson’s Disease: Physiological Aspects and Clinical Implications

“…This rapid differentiation is caused by the block of the two signaling pathways that utilize SMADs for transduction: BMP and TGF-β. Starting from this dual SMAD inhibition, it is possible to generate DA neurons from hESCs, hiPSCs, and adult stem cells and, as recently evidenced, also from perinatal stem cells [20,[34][35][36][37][38][39][40].…”

“…Recently, it has been shown that also human mesenchymal stromal cells (hFM-MSCs) isolated from the amniochorionic membrane [44] can acquire a DA phenotype when treated with the small molecules cocktail used by Kriks et al [20]. In the field of adult stem cells, valproic acid was shown to cooperate in DA differentiation of human spermatogonial stem cells when added to a conditioned medium from olfactory ectomesenchymal cells together with the small molecules SHH, RA, SB431542, FGF8, TGF-β3, and GDNF: the derived DA neurons cells were able to engraft and survive in a PD mouse model, partially rescuing the PD phenotype [34].…”

Local Pattern Transformation Based Feature Extraction for Recognition of Parkinson’s Disease Based on Gait Signals

“…Chiu et al, on the other hand, state that finger motion analysis was used to test the goniometry of finger joints [11]. Based on the survey, the dopaminergic image in the brain is considered as an authentic method for the identification of PD [12]. In gait dynamics, continuous effects of PD were noticed.…”

Neonatal Porcine Germ Cells Dedifferentiate and Display Osteogenic and Pluripotency Properties

“…The resultant cells, known as induced pluripotent stem (iPS) cells, were developed via retroviral delivery of various transcription factors (e.g., Oct3/4, Sox2, c-Myc, and Klf4) to adult dermal fibroblasts [ 18 , 19 , 20 ], hepatocytes, gastric epithelial cells, and even neural stem cells [ 21 , 22 ]. However, the practicality of utilizing different stem cells in clinical settings remains limited due to the ethical and safety concerns over their source [ 23 ]. In addition, current protocols for inducing differentiation into specific cell lineages are inefficient, time-consuming, technically complex, and are not cost effective given the requirement for multiple induction materials [ 24 , 25 ].…”

“…More importantly, we demonstrated that porcine gonocytes are capable of spontaneous dedifferentiation into more primitive stages that express ES cell markers such as SSEA-1, POU5F1, NANOG, and E-cadherin [ 17 ]. This is especially important since in vitro dedifferentiation and trans-differentiation of germ cells in previous reports required coculturing with somatic cells from other species and/or supplementation of the media with multiple exogenous factors [ 7 , 13 , 23 ]. In the present study, we used our recently developed culture conditions to obtain a population of ES-like cells that express pluripotency markers from porcine gonocytes, and then further evaluated their teratoma formation potential using an in vivo implantation assay.…”