Exosomal 2′,3′‐CNP from mesenchymal stem cells promotes hippocampus CA1 neurogenesis/neuritogenesis and contributes to rescue of cognition/learning deficiencies of damaged brain

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.


| INTRODUCTION
The hippocampus CA1 region is essential for cognition, spatial learning, and short-and long-term memory. 1,2 Pyramidal neurons of the hippocampus CA1 region are extremely vulnerable and undergo degeneration in response to many pathological conditions, including ischemia, 3,4 depression, post-traumatic stress disorder, 5 Alzheimer's disease (AD), 6,7 and Parkinson's disease (PD). 8,9 Hippocampal CA1 damage occurring with pathological conditions contributes to memory loss and other cognitive impairments that affect social functioning and daily living of patients. [10][11][12] Adult neurogenesis persists in the mammalian brain throughout life, 13 occurring mainly in two regions: the subventricular zone (SVZ) and the dentate gyrus (DG). 13,14 Additional reports also demonstrated that neurogenesis also occurs in many discrete parts of the adult brain at a low frequency. [15][16][17] Although neurogenesis declines in the adult brain, 18 neurogenesis can be upregulated, albeit transiently, in response to pathological conditions such as ischemia, 19,20 trauma, 21,22 and AD. 23 This induced neurogenesis may be a compensatory response to promote functional recovery of the damaged brain. Since the endogenous regenerative capacity of damaged brain appears to be very limited, 24,25 augmentation of its latent regenerative potential presents a goal as a therapeutic option for CNS diseases. 26 Infusion of growth factors, including epidermal growth factor receptor (EGF), 27 fibroblast growth factor 2 (FGF2), 28 brainderived neurotrophic factor (BDNF), 29 heparin-binding EGF-like growth factor, 30 and vascular endothelial growth factor, 31 can augment regenerative processes such as neuritogenesis and neurogenesis. However, prolonged treatment with growth factors delays and inhibits differentiation of progenitors. 32 Additional research is required to understand how neural progenitors can be recruited to restore specific neuronal cells.
Therapeutic effects of mesenchymal stem cells (MSCs) for neurological diseases such as cerebral infarction, AD, and PD have been reported. 33,34 However, the regenerative effects of MSC transplantation do not result from permanent cell engraftment. Instead, they rely mainly on MSC-produced extracellular components. 35,36 We previously demonstrated that EP 4 antagonist-induced MSC extracellular vesicles (EVs)/exosomes have superior ability to rescue cognition and learning deficiencies caused by hippocampal damage. 37 Consequently, we suspected increases in specific EP 4

antagonist-induced MSC
EVs/exosome cargo components might be responsible for these regenerative effects.
2 0 ,3 0 -Cyclic nucleotide 3 0 -phosphodiesterase (CNP) is highly enriched in myelin but is also present in the cell bodies of oligodendrocytes and neurons. 38,39 Data from CNP transgenic and knockout mice suggest important roles for CNP in myelin formation and axonal integrity. 40,41 CNP proteolysis is increased in aged monkeys, resulting in axonal loss during CNS aging. 42,43 Decreased CNP levels are observed in AD and multiple sclerosis, suggesting CNP loss may contribute to neurological diseases. 44 Here we demonstrate that EVs/exosomes released from EP 4 antagonist-treated MSCs have increased CNP levels. In addition to suppressing astrogliosis and inflammation, 37 systemic administration of EP 4 antagonist-elicited MSC EVs/exosomes promotes neurogenesis and neuritogenesis in damaged hippocampi and can rescue hippocampal CA1 damage-mediated cognition and learning deficiencies. We identify elevated CNP levels as a required component in the EP 4 antagonist-elicited MSC EVs/exosomes to promote neuritogenesis of proliferated progenitors in repairing hippocampal damage, and to restore cognitive, memory, and learning function.
These results expand the possibility of novel neuronal cell regeneration therapies for brain damage and disease.  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.

| EV/exosome isolation
EVs/exosomes were isolated from MSC culture media by differential ultracentrifugation as previously described. 37,45 Briefly, MSCs were treated with DMSO vehicle or 20 μg/mL EP 4 antagonist GW627368X for 4 or 8 days, as indicated in the figure legends (Fig. 5A,B). Culture media were collected and replaced with fresh media supplemented with DMSO or GW627368X every 4 days. The collected culture media were centrifuged at 300g for 5 minutes to remove cells (P1), at 2 000g for 20 minutes (P2), and then at 10 000g for 30 minutes (P3), all at 4 C. Finally, EVs/exosomes (P4) were separated from the supernatant by centrifugation at 110 000g for 60 minutes. The EV/exosome pellet was washed once in phosphate-buffered saline (PBS) and then resuspended in PBS for further analysis and injection.

| Neuron differentiation
NE-4C NSCs were seeded on poly-D-lysine-coated glass slides in six-well dishes. When the NE-4C cells reached 50% confluence, they were treated with PBS, 2 μg/well basal MSC EVs/exosomes, or 2 μg/well GW627368X-induced MSC EVs/exosomes. Treatments were provided every other day for 14 days in the growth medium. The cells were induced to differentiate into neurons by culturing in the growth medium with 5 μM retinoic acid for 8 days, with the medium replaced every 2 days.

| Sphere formation assay
NE-4C cells, which were pretreated as described in Section 2.3, were dissociated into single-cell suspensions and plated in 96 wells of ultralow attachment plates (Corning, New York) at 50 cells/well in DMEM-F12 with 1% methyl cellulose, 1× GlutaMax, penicillin-streptomycin, 20 ng/mL EGF, 20 ng/mL FGF2, and 2% B27. After 6 days, the number of spheres of each well was calculated.

| Sphere staining
Neurospheres were transferred to 12-well plates and washed three times with PBS. After PBS aspiration, the spheres were fixed with of 4% paraformaldehyde (PFA) for 20 minutes at room temperature and then washed three times with PBS. The spheres were blocked by PBS containing 5% normal serum and 0.025% (wt/vol) Triton X-100 for 30 minutes and then subjected to incubation with primary and secondary antibodies at 4 C. The following antibodies were used: anti-

| β3-tubulin polymerization
In vitro β3-tubulin polymerization in EVs/exosomes was analyzed by β3-tubulin polymerization assay (Cytoskeleton, Inc, Denver, Colorado; BK011P), according to the manufacturer's protocol. Twenty micrograms of exosome protein was suspended in 50 μL of buffer 1 from the kit and then subjected to sonication (20 kHz, amplitude 60%) on ice for 20 seconds. Five microliters of exosome lysate/well was loaded in the 96-well assay plate for β3-tubulin polymerization assay.
The β3-tubulin formation in 30 and 60 minutes was quantified by measuring the fluorescence.

| Immunofluorescence studies on cells
Cells on coverslips were fixed in 4% PFA solution in PBS for 15 minutes at room temperature and then washed with PBS. Cells were blocked with 5% bovine serum albumin and 0.3% Triton X-100 in PBS at room temperature for 60 minutes. The coverslips were incubated with anti-β3 tubulin antibodies (Cell Signaling Technology, Danvers, Massachusetts; CS5568; 1:500 dilution) overnight at 4 C and then with secondary antibodies for 1 hour at room temperature.
Cell nuclei were visualized with DAPI. Slides were mounted with Pro-Long Gold Antifade Reagent and imaged using a TCS SP5 II confocal microscope. The confocal images were loaded into ImageJ with NeurphologyJ HT plugin. The number of neurites and total neurite length in the images were calculated using "NeurphologyJ HT" as described. 46

| Animal experiments
All research involving animals complied with protocols approved by the NHRI Committee on Animal Care. B6.CBA-Tg(Camk2a-tTA) and B6.Cg-Tg(tetO-DTA) mice were obtained from Jackson Labs and the National Laboratory Animal Center (NLAC, Taiwan). Camk2a-tTA/ tetO-DTA transgenic mice (Camk2a/DTA mice) express the tetracycline/doxycycline-suppressed transactivator protein (tTA) under control of the hippocampus CA1-specific calcium-calmodulin-dependent kinase II (Camk2a) promoter and diphtheria toxin A (DTA) under the control of a tetracycline/doxycycline-responsive element. Doxycycline region were loaded into ImageJ. The signal-positive area was calculated using "Analyze Particles" under the ImageJ "Analyze" function.
For quantification of CA1 neuron thickness, 8-bit DAPI images covering whole hippocampi were loaded into ImageJ. For each hippocampus, three lines across the DAPI-positive layer of neuronal nuclei in CA1 region were drawn and lengths of the lines were measured using ImageJ. The average length of the three lines represents the CA1 neuron thickness of the hippocampus.

| BrdU incorporation
Camk2a-tTA/tetO-DTA and tetO-DTA mice were labeled with BrdU by i.p. injection daily for 5 days after Dox withdrawal. Three mice per group were used. The mice were weighed before each BrdU injection and were injected with 50 mg BrdU per kg body weight intraperitoneally. After the 5-day injection period, the mice were slowly (20 mL/min) perfused with 20 mL of ice-cold PBS for 3 minutes. Whole brains were excised and fixed with 4% formaldehyde. Paraffin sections of the brains were prepared and stained with anti-BrdU antibody (Abcam, ab6326; 1:250 dilution) overnight at 4 C and then with secondary antibodies (1:300 dilution) for 1 hour at room temperature. Cell nuclei were visualized with DAPI. Slides were mounted with ProLong Gold Antifade Reagent and imaged using a Leica DM2500 microscope.

| Western blotting
Mouse hippocampi were isolated as described. 47 Briefly, the brain was hemisected and the cortical hemisphere was laterally peeled to expose the hippocampus. The hippocampus was laterally removed with a spatula. Hippocampi were ground and lysed with RIPA lysis buffer on ice for 30 minutes for protein extraction.

| Creation of CNP knockdown EVs/exosomes
Expression of CNP was suppressed in MSCs via lentiviral infection as described. 45 The transfection of 293T cells with pLKO-shRNA viral vectors and helper constructs was performed in 10-cm culture dishes.

| CNP assay
One microgram of exosomes were suspended in 50 μL of PBS with HEPES (25 mmol/L) and NaHCO 3 (13 mmol/L) in the presence and absence of 15 μM 2 0 ,3 0 -cAMP and were incubated at 37 C. After 1-hour incubation at 37 C, the mixture was immediately incubated at 100 C for 3 minutes to denature enzymes. The amount of adenosine produced in the 1-hour incubation was measured using the fluorometric adenosine assay, according to the manufacturer's protocol (Biovision [Milpitas, California], #K327); 10 μL of the mixture was diluted with 40 μL of assay buffer and then subjected to the fluorometric adenosine assay. The adenosine produced by the exosomes was measured as adenosine reading (pmole) in the mixture containing only exosomes subtracted from adenosine reading (pmole) in the mixture containing exosomes and 2 0 ,3 0 -cAMP.

| Animal behavior examination
All cognition, learning, and memory tests were performed as described previously. 37 The numbers of animals for each behavioral group are indicated in the figure legends. For each behavioral test, five mice per group were analyzed. Each mouse received only one behavioral test per day. In NORT and NLRT, each mouse was allowed to explore the objects for 5 minutes (exploratory phase) and then was returned to the cage for another 5 minutes. After the 5-minute interval in the cage, the mouse was returned to (a) the chamber with the previously exposed object and a novel object (NORT) or (b) the chamber in which one of the two objects was displaced from its original position (NLRT), for a 3-minute test phase. Exploration counted as positive if the mouse's head was within one inch of the object with neck extended and vibrissae moving. The exploratory phase and test phase were videotaped to measure (time for exploring novel object or location)/ (time for total exploring).
In the MWM test, the learning trials were performed at the same time on day 1 to day 5. The trial began from a different quadrant of the pool for each day (second and third quadrant for day 2, third and fourth quadrant for day 3, first and fourth quadrant for day 4, and first and second quadrant for day 5). Each trial ended when the mouse arrived at the platform, or after 60 seconds had passed. Mice were immediately removed from the pool at the end of the trial. All tracks from all trials were recorded and analyzed using the Videotrack software (Viewpoint).
For Figure 7D, two-way analysis of variance was used to analyze the difference of the latency between groups and a P value ≤.05 was considered statistically significant.

| EP 4 antagonist-induced MSC EVs/exosomes promote the formation of neurospheres in cell culture
We previously showed that EP 4 antagonist-induced MSC EVs/exosomes (GW MSC EVs/exosomes) have suppressive effects on both reactive astrocytes and active microglia; these suppressive activities contribute to the regenerative effects of the GW MSC EVs/exosomes on damaged brains. 37 Here we further investigate whether the effects of EP 4 antagonist-induced MSC EVs/ exosomes on damaged brains also act on neuronal precursors and neurons. As described previously, bone marrow MSCs were characterized by their surface marker profiles and their abilities to differentiate into adipocytes and osteocytes before being subjected to EV and GW EP 4 antagonist-induced MSC EVs/exosome (GWEV) collection. 37 Four-day pretreatment with GWEVs significantly increased the neurosphere-forming ability of mouse NE-4C NSCs, whereas pretreatment with basal MSC EVs did not increase neurosphere formation ( Figure 1A; Figure S1A). The spheres were analyzed for Nestin and GFAP expression to confirm their identity as neurospheres. For spheres in all the three groups, we observed that most cells in the spheres expressed Nestin and only a few sphere cells expressed GFAP ( Figure S1B). The characteristics of these spheres correspond with that of neurospheres described in the literature. [48][49][50][51][52][53] 3.2 | EP 4 antagonist-induced MSC EVs/exosomes promote neuritogenesis in cell culture β3-tubulin is the main structural protein of neuron cytoskeletal microtubules in neurites; β3-tubulin polymerization can be used as a functional marker for assessing neuritogenesis. 54 (Figure 2A,B) (see Section 2). We previously compared administration of EVs/exosomes by intracardiac injection, tail vein injection, and intraorbital injection and observed optimal delivery of EVs/exosomes to brain by intracardiac injection. 37 Consequently, in our subsequent experiments, EVs/exosomes and PBS were administered to the mice via intracardiac injection. PBS, MSC EVs/exosomes (EV), or EP 4 antagonist-induced MSC GWEVs/exosomes (GWEV) were administered twice, by intracardiac injection at the time points indicted in Figure 2C, to mice with damaged hippocampi.

| EP 4 antagonist-elicited MSC EVs/exosomes increase CA1 neurons in the damaged hippocampus
In the hippocampus, the CA1 region contains a unique, compact layer of pyramidal neurons consisting of approximately eight rows of neuron bodies ( Figure 2B, UC). 55 The number of hippocampal CA1 pyramidal neurons is positively correlated with the thickness of the CA1 pyramidal cell body layer. 56 We demonstrated above that hippocampal damage decreased this neuron layer in hippocampal CA1 of   MAP2 is located mainly in neurites and binds to β3-tubulin to stabilize microtubule growth. 59 Recovery of the functional state of neurons after damage is associated with increased MAP2 production. 60 Hippocampal MAP2 expression was analyzed using immunostaining.  Figure 4B) or from EP 4 antagonist-treated MSCs (lower panels, Figure 4B). Although DCX levels were very sparse in the CA1 region of both UC and DC hippocampi (upper panels, Figure 4B), elevated DCX levels were present in the hippocampal CA1 regions of both MSC EVinjected mice (middle panels, Figure 4B) and MSC GWEV-injected mice (lower panels, Figure 4B

Elimination of individual cargo proteins from MSCs into MSC
EVs/exosomes provides an opportunity to determine the roles of these components in facilitating recovery, both for hippocampal functional properties (eg, cognition, learning, and memory) and for correlative studies in cellular and physical properties (eg, astrogliosis, inflammation, blood brain barrier properties, neuritogenesis, neuronal recovery).

CNP levels in the EP 4 antagonist-elicited MSC GWEVs/exosomes
were elevated about 20-fold compared with that of basal MSC EV/exosomes ( Figure 5B). Forty percent of CNP associates with lipid rafts. 69 Lipid raft-associated CNP decreases with age and the agerelated change likely alters the function of CNP for axonal maintenance in monkeys. 43,73 CNP knockout mice have structurally normal myelin but demonstrate neurite degradation and neurodegeneration, 40 suggesting CNP contributes to maintenance of neurite integrity.
To investigate the contribution of elevated CNP in EP 4 antagonist-elicited MSC EVs/exosomes to stimulated activities for deficits in the damaged hippocampus, we used shRNA reduction of CNP protein in MSCs to prepare GWEVs/exosomes deficient in CNP.
We then compared the regenerative efficacy of MSC GWEVs/ exosomes elicited from control and from CNP knockdown MSC cells.

| CNP in EP
As an MAP, CNP binds tubulin multimers and induces microtubule polymerization. 77 In this manner, CNP directs the formation of branched process outgrowth in glia and neuritogenesis in neurons.
We investigated whether exosomal CNP contributes to the ability of EP 4 antagonist-induced MSC GWEVs/exosomes to increase β3-tubulin polymerization. Although EP 4 antagonist-induced MSC GWEVs/exosomes increased β3-tubulin polymerization, knocking down CNP in the EP 4 antagonist-induced MSC EVs/exosomes decreased the ability of these exosomes to promote β3-tubulin polymerization to the level of basal MSC EVs/exosomes ( Figure 5F,G), suggesting that the enriched CNP in EP 4 antagonist-induced MSC EVs/exosomes contributes to the GWEV/exosome elicited β3-tubulin polymerization required in neuritogenesis.
3.9 | Exosomal CNP contributes to EP 4 antagonistinduced MSC GWEV/exosome promoted neuritogenesis of CA1 neurons in the damaged hippocampus Mice with undamaged hippocampi (UC) and Camk2a/DTA mice with damaged hippocampi (DC) that were injected with MSC GWEVs/ exosomes prepared from cells expressing either shGFP or shcontrol rapidly and progressively found the platform in successive trials in MWM tests at the 3rd, 4th, and 5th day of training ( Figure 7D and Figure S6C).
In contrast, the Camk2a/DTA mice injected with PBS (DC) or GWEVs/ exosomes prepared from MSCs expressing either shCNP-1 or shCNP-2 failed to find the platform over the course of the experiments ( Figure 7D and Figure S6C), demonstrating that the elevated CNP present in EP 4 antagonist-elicited MSC EVs/exosomes is required for the therapeutic capability exhibited by MSC GWEVs/exosomes for this spatial navigation and memory deficiency analysis. In summary, the data suggest that MSC GWEV/exosomal CNP is necessary for the therapeutic potential of EP 4 antagonist-elicited MSC EVs/exosomes on memory and learning deficiencies in mice with damaged hippocampi.
It should be emphasized that it is likely that many other components of the MSC GWEVs/exosomes are also required for the therapeutic efficacies demonstrated here. Elimination of other proteinsand perhaps RNA molecules and small molecule metabolites or signaling molecules-in the MSC GWEVs/exosomes will allow identification of components that are essential both for normal memory and learning processes and for therapeutic efficacy of MSC GWEVs/exosomes. Systematic evaluation of these components may identify a hierarchy of such molecules for therapeutic efficacy as well as cell-type specific restoration/regeneration responses for distinct GWEV/exosome cargo constituents.

| DISCUSSION
Many studies suggest paracrine signaling as the primary mechanism of MSC action, since administration of MSC-secreted molecules can often convey the biologic effects of MSCs. 79,80 Using MSC-secreted molecules, including -derived EVs/exosomes, for therapy does not cause many of the difficulties observed with MSC-based therapies, including complications of cell implantation, ectopic tissue formation, or unwanted engraftment; consequently, the use of MSC-derived EVs/exosomes for therapy may attenuate many of the safety concerns related to the use of living stem cells. The therapeutic potential of basal MSC-derived EV/exosomes, released in normal MSC culture conditions, for damaged brains has recently been explored by several groups. [81][82][83] However, we found that the basal MSC EVs/exosomes did not substantially suppress astrogliosis or inflammation, 37 did not promote neuritogenesis (Figures 1-4), and did not restore cognition, memory, or learning in the damaged hippocampi. In contrast, EP 4 antagonist-induced MSC EVs/exosomes suppressed astrogliosis and inflammation, 37 promoted neurogenesis and neuritogenesis ( Figures 1-4), and restored cognition, memory, and learning in the damaged hippocampi. 37 We demonstrated that EP 4 antagonistinduced MSC EVs/exosomes can rescue the deficiencies of cognition, memory, and learning caused by the damage in hippocampus CA1, whereas the basal MSC EVs/exosomes could not rescue this brain dysfunction caused by the damage in the hippocampus. We concluded that EP 4 antagonist-induced MSC EVs/exosomes have superior regenerative effects on various aspects of damaged brain, compared with basal MSC EVs/exosomes. 37 During development, pyramidal neurons in the CA1 region are generated from progenitors migrating from the ventricular zone and the SVZ. 84 Although hippocampal pyramidal neurons are generated primarily during embryonic development, several studies demonstrate that brain injury and disease reactivate this process in the adult. 19,20,85 Adult hippocampal neurogenesis is implicated in various cognitive and emotional processing abilities. 78,86 The generation of new CA1 neurons is associated with a restoration of learning and memory functions. 78 Figure 5A). Because we previously demonstrated, in mammary stem cells, that blocking PGE 2 /EP 4 signaling promotes protein association with lipid rafts and preferentially increases sorting of these proteins into released EVs/exosomes, 45 we suspected that EP 4 antagonism might also increase sorting of lipid raftassociated proteins into MSC EVs/exosomes and that preferentially sorted proteins in EP 4 antagonist-induced MSC EVs/exosomes might include components necessary for the superior regenerative potential of the EVs/exosomes on brain damage.
To initiate an investigation of functional roles of proteins in EP 4 antagonist-elicited EVs/exosomes, we decided to eliminate candidate proteins in the MSCs by shRNA knockdown, produce EP 4 -induced GW EVs/exosomes from these cells, and test the resultant GW EVs/exosomes in their restorative ability for cell-specific functions and for recovery of learning, cognition, and memory in mice with damaged hippocampi. We begin this study by selecting CNP, an EV/exosome component that is enriched in GW antagonist-elicited EVs/exosomes compared with basal EV/exosomes ( Figure 5B). CNP supports neurite integrity by promoting neurite cytoskeletal β3-tubulin polymerization at low molar ratios and by converting toxic 2 0 ,3 0 -cAMP and producing neuroprotective adenosine. 40,68,88 2 0 ,3 0 -cAMP, derived from mRNA degradation in damaged tissue, 89 can increase mitochondrial permeability; the increased permeability leads to apoptosis and necrosis of neural cells. 90,91 CNP can decrease toxic 2 0 ,3 0 -cAMP and produce neuroprotective adenosine, 75 which can suppress reactive astrogliosis by limiting excessive astrocyte proliferation 76 and act as an axonal protectant 92 in damaged brains. CNP −/− knockout mice develop axonal degradation with age while their myelin is normal in structure and exhibit both motor and memory defects. 40,93 CNP is highly enriched in EP 4 antagonist-elicited MSC EVs/exosomes ( Figure 5B). Reduction of CNP in EP 4 antagonistelicited MSC EVs/exosomes derived from MSCs in which CNP has been knocked down by the appropriate shRNAs (shCNP-1 and shCNP-2) decreases the ability of these exosomes to convert toxic 2 0 ,3 0 -cAMP to neuroprotective adenosine in vitro ( Figure 5E), to promote neurite cytoskeletal β3-tubulin polymerization in vitro ( Figure 5F,G), to promote neuritogenesis/neurogenesis (Figure 6), and to recover CNS memory, learning, and cognition functions (Figure 7).
These results suggest that the presence of elevated CNP as cargo is required for the regenerative/therapeutic efficacy of EP 4 antagonistelicited MSC EVs/exosomes. Decreases in CNP expression have been linked to AD and Down's syndrome. 44 BDNF increases CNP in the brains and improves functional recovery and connectivity of animals with ischemic stroke. 94 We observed that, in the EP 4 antagonistelicited MSC EV/exosome-treated damaged brains, the induced DCXpositive neuronal precursor cells contained CNP protein ( Figure S3).
These observations support the suggestion that the increased CNP present in EP 4 antagonist-elicited MSC EVs/exosomes may contribute to their increased therapeutic efficacy in restoration of function in the damaged brains.
Here we provide evidence that targeted deletion of MSC components prior to eliciting EP 4 antagonist-induced MSC EVs/exosomes can identify individual components that restore distinct behavioral, biochemical, and cellular hippocampal properties. In this way, we show that the elevated CNP in EP 4 antagonist-induced MSC EVs/exosomes is necessary for their ability to promote neuritogenesis and neurogenesis in damaged brain and for functional recovery of memory, cognition, and learning in an experimental paradigm. Elevated levels of IL-2, IL-10, N-cad, CD44, CD90, and CD109 are also present in EP 4 antagonist-induced MSC EV/exosomes ( Figure 5A). 37 The presence of MSC EV/exosome cargo for cell targeting (eg, for wounded tissues 95 ), suppressing inflammation, or blood-brain barrier restoration can also be examined by reduction of candidate cargo components in MSCs followed by EV/exosome production and testing in appropriate animal models.

CONFLICT OF INTEREST
The authors indicated no potential conflicts of interest. conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, financial support, administrative support, final approval of manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of this article.
How to cite this article: Chen S-Y, Lin M-c, Tsai J-S, et al.