Click chemistry extracellular vesicle/peptide/chemokine nanocarriers for treating central nervous system injuries.

Central nervous system (CNS) injuries, including stroke, traumatic brain injury, and spinal cord injury, are essential causes of death and long-term disability and are difficult to cure, mainly due to the limited neuron regeneration and the glial scar formation. Herein, we apply extracellular vesicles (EVs) secreted by M2 microglia to improve the differentiation of neural stem cells (NSCs) at the injured site, and simultaneously modify them with the injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF-1) on their surface via copper-free click chemistry to recruit NSCs, inducing their neuronal differentiation, and serving as the nanocarriers at the injured site (Dual-EV). Results prove that the Dual-EV could target human umbilical vascular endothelial cells (HUVECs), recruit NSCs, and promote the neuronal differentiation of NSCs in vitro. Furthermore, 10 miRNAs are found to be upregulated in Dual-M2-EVs compared to Dual-M0-EVs via bioinformatic analysis, and further NSC differentiation experiment by flow cytometry reveals that among these miRNAs, miR30b-3p, miR-222-3p, miR-129-5p, and miR-155-5p may exert effect of inducing NSC to differentiate into neurons. In vivo experiments show that Dual-EV nanocarriers achieve improved accumulation in the ischemic area of stroke model mice, potentiate NSCs recruitment, and increase neurogenesis. This work provides new insights for the treatment of neuronal regeneration after CNS injuries as well as endogenous stem cells, and the click chemistry EV/peptide/chemokine and related nanocarriers for improving human health.

Brain Microenvironment Responsive and Pro-Angiogenic Extracellular Vesicle-Hydrogel for Promoting Neurobehavioral Recovery in Type 2 Diabetic Mice After Stroke.

Stroke patients with diabetes have worse neurological outcomes than non-diabetic stroke patients, and treatments beneficial for non-diabetic stroke patients are not necessarily effective for diabetic stroke patients. While stem cell-derived extracellular vesicles (EVs) show potential for treating stroke, the results remain unsatisfactory due to the lack of approaches for retaining and controlling EVs released into the brain. Herein, a glucose/reactive oxygen species dual-responsive hydrogel showing excellent injectability, biocompatibility, and self-healing capability is introduced as an EVs-loading vehicle and an intelligent EVs sustained releasing system in the brain. These EVs-hydrogels are developed via crosslinking of phenylboronic acid-modified hyaluronic acid and Poly vinyl alcohol, and fusion with neural stem cell-derived EVs. The results show EVs are stably incorporated into the hydrogels and can be controllably released in response to the brain microenvironment after stroke in type 2 diabetic mice. The EVs-hydrogels exert an excellent angiogenic effect, increasing the migration and tube formation of human umbilical vein endothelial cells. In addition, injection of EVs-hydrogels into the ischemic mouse brain enhances EVs retention and facilitates sustained release, promotes angiogenesis, and improves neurobehavioral recovery. These results suggest such a microenvironment responsive and sustained release EVs-hydrogel system offers a safe, and efficient therapy for diabetic stroke.

Targeted delivery of fat extract by platelet membrane-cloaked nanocarriers for the treatment of ischemic stroke.

BACKGROUND: Our previous studies suggest that human fat extract (FE) contains a variety of angiogenic factors and may provide an alternative treatment option for stroke. However, the therapeutic effect is largely limited due to its short half-life, and inaccurate targeting. RESULTS: Herein, we leverage the targeting abilities of platelets (PLTs) to the lesion area of stroke and Arg-Gly-Asp (RGD) peptides to the angiogenic blood vessels to develop a biomimetic nanocarrier that capable of delivering FE precisely to treat stroke. The biomimetic nanocarriers are comprised of FE-encapsulated PLGA (poly(lactic-co-glycolic acid)) core enclosed by RGD peptides decorated plasma membrane of PLTs, namely RGD-PLT@PLGA-FE. We found that RGD-PLT@PLGA-FE not only targeted damaged and inflamed blood vessels but also achieved rapid accumulation in the lesion area of ischemic brain. In addition, RGD-PLT@PLGA-FE kept a sustained release behavior of FE at the lesion site, effectively increased its half-life and promoted angiogenesis and neurogenesis with delivering neurotrophic factors including BDNF, GDNF and bFGF to the brain, that ultimately resulted in blood flow increase and neurobehavioral recovery. CONCLUSIONS: In conclusion, our study provides a new strategy to design a biomimetic system for FE delivery and it is a promising modality for stroke therapy.

M2 microglia-derived extracellular vesicles promote white matter repair and functional recovery via miR-23a-5p after cerebral ischemia in mice.

Rationale: White matter repair is critical for the cognitive and neurological functional recovery after ischemic stroke. M2 microglia are well-documented to enhance remyelination and their extracellular vesicles (EVs) mediate cellular function after brain injury. However, whether M2 microglia-derived EVs could promote white matter repair after cerebral ischemia and its underlying mechanism are largely unknown. Methods: EVs were isolated from IL-4 treated microglia (M2-EVs) and untreated microglia (M0-EVs). Adult ICR mice subjected to 90-minute transient middle cerebral artery occlusion received intravenous EVs treatment for seven consecutive days. Brain atrophy volume, neurobehavioral tests were examined within 28 days following ischemia. Immunohistochemistry, myelin transmission electron microscope and compound action potential measurement were performed to assess white matter structural remodeling, functional repair and oligodendrogenesis. The effects of M2-EVs on oligodendrocyte precursor cells (OPCs) were also examined in vitro. EVs' miRNA sequencing, specific miR-23a-5p knockdown in M2-EVs and luciferase reporter assay were used to explore the underlying mechanism. Results: M2-EVs reduced brain atrophy volume, promoted functional recovery, oligodendrogenesis and white matter repair in vivo, increased OPC proliferation, survival and differentiation in vitro. miR-23a-5p was enriched in M2-EVs and could promote OPC proliferation, survival and maturation, while knocking down miR-23a-5p in M2-EVs reversed the beneficial effects of M2-EVs both in vitro and in vivo. Luciferase reporter assay showed that miR-23a-5p directly targeted Olig3. Conclusion: Our results demonstrated that M2 microglia could communicate to OPCs through M2-EVs and promote white matter repair via miR-23a-5p possibly by directly targeting Olig3 after ischemic stroke, suggesting M2-EVs is a novel and promising therapeutic strategy for white matter repair in stroke and demyelinating disease.

Extracellular vesicles from adipose-derived stem cells promote microglia M2 polarization and neurological recovery in a mouse model of transient middle cerebral artery occlusion.

BACKGROUND: Adipose-derived stem cells (ADSCs) and their extracellular vesicles (EVs) have therapeutic potential in ischemic brain injury, but the underlying mechanism is poorly understood. The current study aimed to explore the contribution of miRNAs in ADSC-EVs to the treatment of cerebral ischemia. METHODS: After the intravenous injection of ADSC-EVs, therapeutic efficacy was evaluated by neurobehavioral tests and brain atrophy volume. The polarization of microglia was assessed by immunostaining and qPCR. We further performed miRNA sequencing of ADSC-EVs and analyzed the relationship between the upregulated miRNAs in ADSC-EVs and microglial polarization-related proteins using Ingenuity Pathway Analysis (IPA). RESULTS: The results showed that ADSC-EVs reduced brain atrophy volume, improved neuromotor and cognitive functions after mouse ischemic stroke. The loss of oligodendrocytes was attenuated after ADSC-EVs injection. The number of blood vessels, as well as newly proliferated endothelial cells in the peri-ischemia area were higher in the ADSC-EVs treated group than that in the PBS group. In addition, ADSC-EVs regulated the polarization of microglia, resulting in increased repair-promoting M2 phenotype and decreased pro-inflammatory M1 phenotype. Finally, STAT1 and PTEN were highlighted as two downstream targets of up-regulated miRNAs in ADSC-EVs among 85 microglia/macrophage polarization related proteins by IPA. The inhibition of STAT1 and PTEN by ADSC-EVs were confirmed in cultured microglia. CONCLUSIONS: In summary, ADSC-EVs reduced ischemic brain injury, which was associated with the regulation of microglial polarization. miRNAs in ADSC-EVs partly contributed to their function in regulating microglial polarization by targeting PTEN and STAT1.

Plasma from healthy donors protects blood-brain barrier integrity via FGF21 and improves the recovery in a mouse model of cerebral ischaemia.

BACKGROUND: Healthy plasma therapy reverses cognitive deficits and promotes neuroplasticity in ageing brain disease. However, whether healthy plasma therapy improve blood-brain barrier integrity after stroke remains unknown. METHODS: Here, we intravenously injected healthy female mouse plasma into adult female ischaemic stroke C57BL/6 mouse induced by 90 min transient middle cerebral artery occlusion for eight consecutive days. Infarct volume, brain atrophy and neurobehavioural tests were examined to assess the outcomes of plasma treatment. Cell apoptosis, blood-brain barrier integrity and fibroblast growth factor 21 knockout mice were used to explore the underlying mechanism. RESULTS: Plasma injection improved neurobehavioural recovery and decreased infarct volume, brain oedema and atrophy after stroke. Immunostaining showed that the number of transferase dUTP nick end labelling+/NeuN+ cells decreased in the plasma-injected group. Meanwhile, plasma injection reduced ZO-1, occluding and claudin-5 tight junction gap formation and IgG extravasation at 3 days after ischaemic stroke. Western blot results showed that the FGF21 expression increased in the plasma-injected mice. However, using FGF21 knockout mouse plasma injecting to the ischaemic wild-type mice diminished the neuroprotective effects. CONCLUSIONS: Our study demonstrated that healthy adult plasma treatment protected the structural and functional integrity of blood-brain barrier, reduced neuronal apoptosis and improved functional recovery via FGF21, opening a new avenue for ischaemic stroke therapy.

Natural medicine in neuroprotection for ischemic stroke: Challenges and prospective.

There is a growing interest in using natural medicines to treat stroke as the leading cause of disability worldwide. Especially plant-derived natural medicines have multiple beneficial effects, including antioxidative, anti-inflammatory, antiapoptotic, suggesting their potential for stroke treatment. However, failure in clinical translation has created a substantial challenge for their use as stroke treatment. This review summarises the status of using natural medicinal formulations in stroke treatment, as well as the challenges. Although natural medicines have excellent prospects for ischaemic stroke treatment, several concerns need to be addressed for translating the success in animal research to humans. Specifically, there should be more considerations for investigating the precise mechanisms of natural medicines mediating the neuroprotective effects on stroke. Moreover, to further improve the prospects of clinical translation, further studies should be performed in aged animals rather than young adult animals.

Internalization of the TAT-PBX1 fusion protein significantly enhances the proliferation of human hair follicle-derived mesenchymal stem cells and delays their senescence.

OBJECTIVES: To express a TAT-PBX1 fusion protein using a prokaryotic expression system and to explore potential effects of TAT-PBX1 in the proliferation and senescence of human hair follicle-derived mesenchymal stem cells. RESULTS: The TAT-PBX1 fusion was produced in inclusion bodies and heterogenously expressed in Rosetta (DE3) cells. Immunofluorescence staining showed that TAT-PBX1 fusion proteins were internalized by human hair follicle-derived mesenchymal stem cells. The growth rate of cells was increased after treatment with more than 5.0 µg/mL of TAT-PBX1. The rate of senescence-associated ß-galactosidase positive cells was reduced in the 10.0 µg/mL TAT-PBX1 group (28%) than the 0 µg/mL control group (60%). Cells treated with the TAT-PBX1 fusion protein showed higher expression of p-AKT (1.22-fold that of the control), which indicates that TAT-PBX1 activated AKT pathway after cellular uptake. CONCLUSIONS: The TAT-PBX1 fusion protein increased the proliferation of hair follicle mesenchymal stem cells and delayed their senescence by activating the AKT pathway following internalization by cells.

PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis.

BACKGROUND: PBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3ß pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs. METHODS: A lentiviral vector designed to carry the PBX1 sequence or PBX1 short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of the NANOG promoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT. RESULTS: Overexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of ß-catenin, resulting in the progression of the cell cycle from G0/G1 to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3ß pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency. CONCLUSIONS: PBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3ß signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3ß pathway during the early stages of reprogramming.

Epidermal Growth Factor Induces Proliferation of Hair Follicle-Derived Mesenchymal Stem Cells Through Epidermal Growth Factor Receptor-Mediated Activation of ERK and AKT Signaling Pathways Associated with Upregulation of Cyclin D1 and Downregulation of p16.

The maintenance of highly proliferative capacity and full differentiation potential is a necessary step in the initiation of stem cell-based regenerative medicine. Our recent study showed that epidermal growth factor (EGF) significantly enhanced hair follicle-derived mesenchymal stem cell (HF-MSC) proliferation while maintaining the multilineage differentiation potentials. However, the underlying mechanism remains unclear. Herein, we investigated the role of EGF in HF-MSC proliferation. HF-MSCs were isolated and cultured with or without EGF. Immunofluorescence staining, flow cytometry, cytochemistry, and western blotting were used to assess proliferation, cell signaling pathways related to the EGF receptor (EGFR), and cell cycle progression. HF-MSCs exhibited surface markers of mesenchymal stem cells and displayed trilineage differentiation potentials toward adipocytes, chondrocytes, and osteoblasts. EGF significantly increased HF-MSC proliferation as well as EGFR, ERK1/2, and AKT phosphorylation (p-EGFR, p-ERK1/2, and p-AKT) in a time- and dose-dependent manner, but not STAT3 phosphorylation. EGFR inhibitor (AG1478), PI3K-AKT inhibitor (LY294002), ERK inhibitor (U0126), and STAT3 inhibitor (STA-21) significantly blocked EGF-induced HF-MSC proliferation. Moreover, AG1478, LY294002, and U0126 significantly decreased p-EGFR, p-AKT, and p-ERK1/2 expression. EGF shifted HF-MSCs at the G1 phase to the S and G2 phase. Concomitantly, cyclinD1, phosphorylated Rb, and E2F1expression increased, while that of p16 decreased. In conclusion, EGF induces HF-MSC proliferation through the EGFR/ERK and AKT pathways, but not through STAT-3. The G1/S transition was stimulated by upregulation of cyclinD1 and inhibition of p16 expression.

Feasibility of human hair follicle-derived mesenchymal stem cells/CultiSpher(®)-G constructs in regenerative medicine.

The use of human mesenchymal stem cells (hMSCs) in cell therapies has increased the demand for strategies that allow efficient cell scale-up. Preliminary data on the three-dimensional (3D) spinner culture describing the potential use of microcarriers for hMSCs culture scale-up have been reported. We exploited a rich source of autologous stem cells (human hair follicle) and demonstrated the robust in vitro long-term expansion of human hair follicle-derived mesenchymal stem cells (hHF-MSCs) by using CultiSpher(®)-G microcarriers. We analyzed the feasibility of 3D culture by using hHF-MSCs/CultiSpher(®)-G microcarrier constructs for its potential applicability in regenerative medicine by comparatively analyzing the performance of hHF-MSCs adhered to the CultiSpher(®)-G microspheres in 3D spinner culture and those grown on the gelatin-coated plastic dishes (2D culture), using various assays. We showed that the hHF-MSCs seeded at various densities quickly adhered to and proliferated well on the microspheres, thus generating at least hundreds of millions of hHF-MSCs on 1 g of CultiSpher(®)-G within 12 days. This resulted in a cumulative cell expansion of greater than 26-fold. Notably, the maximum and average proliferation rates in 3D culture were significantly greater than that of the 2D culture. However, the hHF-MSCs from both the cultures retained surface marker and nestin expression, proliferation capacity and differentiation potentials toward adipocytes, osteoblasts and smooth muscle cells and showed no significant differences as evidenced by Edu incorporation, cell cycle, colony formation, apoptosis, biochemical quantification and qPCR assays.

Cyclosporine A increases hair follicle growth by suppressing apoptosis-inducing factor nuclear translocation: a new mechanism.

Cyclosporine A (CsA) enhances hair growth through caspase-dependent pathways by retarding anagen-to-catagen phase transition in the hair follicle growth cycle. Whether apoptosis-inducing factor (AIF), a protein that induces caspase-independent apoptosis, can regulate the hair follicle cycle in response to CsA is currently unclear. Here, we show that the pro-hair growth properties of CsA are in part due to blockage of AIF nuclear translocation. We first isolate hair follicles from murine dorsal skin. We then used Western blot, immunohistochemistry and immunofluorescence to evaluate the expression and localization of AIF in hair follicles. We also determined whether modulation of AIF was responsible for the effects of CsA at the anagen-to-catagen transition. AIF was expressed in hair follicles during the anagen, catagen and telogen phases. There was significant nuclear translocation of AIF as hair follicles transitioned from anagen to late catagen phase; this was inhibited by CsA, likely due to reduced cyclophilin A expression and attenuated AIF release from mitochondria. However, we note that AIF translocation was not completely eliminated, which likely explains why the transition to catagen phase was severely retarded by CsA, rather than being completely inhibited. We speculate that blockade of the AIF signalling pathway is a critical event required for CsA-dependent promotion of hair growth in mice. The study of AIF-related signalling pathways may provide insight into hair diseases and suggest potential novel therapeutic strategies.