Generation of Functional Dopaminergic Neurons from Human Spermatogonial Stem Cells to Rescue Parkinsonian Phenotypes

Abstract

Background: Recent progress in the induced generation of dopaminergic neurons (DANs) 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 treatment of human neurological disorders. Additionally, the current protocols for in vitro differentiation or cell reprogramming to generate human DANs are laborious, time-consuming and expensive, and efficient conversion of human spermatogonial stem cells (hSSCs) to functional DANs has not yet been achieved. Methods: Primary hSSCs from 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 DAN 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 DANs and genuine ones. Concomitantly, the functional properties of converted DANs including synapse formation, dopamine release, electrophysiological activity, and neuron specific Ca2 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 MTPT-induced PD animals. Findings: The hSSCs-derived neurons not only acquired neuronal morphological features but also expressed various phenotypic genes and proteins characteristic of DANs and several effectors critical for pro-DA neurogenesis. Strikingly, as the period of induction was prolonged, expression of the critical molecules for DAN epigenetic status gradually increased while SSC-specific markers sharply decreased. After 3 weeks of induction, the transdifferentiation efficiency reached 21%. In addition, hierarchical clustering analysis showed that the differentiated DANs closely resembled genuine ones. Furthermore, the hSSC-derived neurons gained sophisticated functional properties of wild-type DANs, and pro-induced hSSCs efficiently survived, migrated, and differentiated into DANs without tumorigenesis after transplantation into mouse striatum, leading to improvement of functional deficits in PD animals. Interpretation: Neurons-derived from hSSCs acquired DAN morphological features and functional properties, and rescued Parkinsonian phenotypes. Our strategy for the conversion of hSSCs into DANs is very efficient and thus may provide an alternative approach suitable for clinical cell therapy to treat neurodegenerative diseases including PD. Funding Statement: This work was supported by the Natural Science Foundation of China (grant nos. 81371411, 81571208, 81472098 and 81772357), the Natural Science Foundation of Shaanxi province (2017JM8012), Shanghai Jiaotong University Med-X Fund (No.YG2014MS45), and a key fund of Honghui Hospital (YJ2017001). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Human testicular tissues were obtained from clinically obstructive azoospermic (OA) patients who had normal spermatogenesis and all corpse subjects for autopsy and histopathological assessment in accordance with local and Nation Ethics committee approved. All animal procedures were performed in agreement with the National Institutes of Health guidelines and were approved by the Institutional Ethical Review Committee of Hong Hui Hospital affiliated by Xi’an Jiaotong University.