Exosomal 2′,3′-CNP from mesenchymal stem cells promotes hippocampus CA1 neurogenesis/neuritogenesis and contributes to rescue of cognition/learning deficiencies of damaged brain
Authors: Chen Shih-Yin , Lin Meng-chieh , Tsai Jia-Shiuan , He Pei-Lin , Luo Wen-Ting , Chiu Ing-Ming , Herschman Harvey R. , Li Hua-Jung 
Published: January 2020

Abstract
Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC-derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist-induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist-induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2′,3′-Cyclic nucleotide 3′-phosphodiesterase (CNP) levels in EP4 antagonist-induced MSC EVs/exosomes are 20-fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist-induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting β3-tubulin polymerization and in converting toxic 2′,3′-cAMP into neuroprotective adenosine. CNP-depleted EP4 antagonist-induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4-antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP-depleted EP4 antagonist-induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist-elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses.

Significance statement Mesenchymal stem cells (MSCs) have therapeutic effects for neurological diseases and damage; however, their therapeutic effects are mediated by components of the MSC secretome. Compared with basal MSC EVs/exosomes, EP4 antagonist-induced MSC EVs/exosomes have superior regenerative ability to rescue damaged brain functions. Elevated CNP levels are required for the enhanced ability of EP4-induced MSC EVs/exosomes to promote β3-tubulin polymerization, decrease toxic 2′,3′-cAMP, produce neuroprotective adenosine, induce neurogenesis, and elicit neuritogenesis in damaged hippocampi. At a functional level, elevated EV/exosome CNP levels are required to rescue cognition and learning deficiencies caused by this damage. These data suggest that CNP modulation is a potential target for treating brain damage and neural degeneration diseases. Moreover, these results suggest a generalized approach to identifying causal roles for EV/exosome cargo components in a variety of regenerative applications.