Mesenchymal Stem Cells Membrane Research Articles

Reduction of Oxidative Stress and Excitotoxicity by Mesenchymal Stem Cell Biomimetic Co‐Delivery System for Cerebral Ischemia‐Reperfusion Injury Treatment

AbstractA cerebral ischemia‐reperfusion injury is ensued by an intricate interplay between various pathological processes including excitotoxicity, oxidative stress, inflammation, and apoptosis. For a long time, drug intervention policies targeting a single signaling pathway have failed to achieve the anticipated clinical efficacy in the intricate and dynamic inflammatory environment of the brain. Moreover, inadequate targeted drug delivery remains a significant challenge in cerebral ischemia‐reperfusion injury therapy. In this study, a multifunctional nanoplatform (designated as PB‐006@MSC) is developed using ZL006‐loaded Prussian blue nanoparticles (PBNPs) camouflaged by a mesenchymal stem cell (MSC) membrane (MSCm). ZL006 is a neuroprotectant. It can be loaded efficiently into the free radical scavenger PBNP through mesoporous adsorption. This can simultaneously modulate multiple targets and pathways. MSCm biomimetics can reduce the nanoparticle immunogenicity, efficiently enhance their homing capability to the cerebral ischemic penumbra, and realize active‐targeting therapy for ischemic stroke. In animal experiments, PB‐006@MSC integrated reactive oxygen species (ROS) scavenging and neuroprotection. Thereby, it selectively targeted the cerebral ischemic penumbra (about fourfold higher accumulation at 24 h than in the non‐targeted group), demonstrated a remarkable therapeutic efficacy in reducing the volume of cerebral infarction (from 37.1% to 2.3%), protected the neurogenic functions, and ameliorated the mortality.

Precisely targeted drug delivery by mesenchymal stem cells-based biomimetic liposomes to cerebral ischemia-reperfusion injured hemisphere

The precise and targeted delivery of therapeutic agents to the lesion sites remains a major challenge in treating brain diseases represented by ischemic stroke. Herein, we modified liposomes with mesenchymal stem cells (MSC) membrane to construct biomimetic liposomes, termed MSCsome. MSCsome (115.99 ± 4.03 nm) exhibited concentrated accumulation in the cerebral infarcted hemisphere of mice with cerebral ischemia-reperfusion injury, while showing uniform distribution in the two cerebral hemispheres of normal mice. Moreover, MSCsome exhibited high colocalization with damaged nerve cells in the infarcted hemisphere, highlighting its advantageous precise targeting capabilities over liposomes at both the tissue and cellular levels. Leveraging its superior targeting properties, MSCsome effectively delivered Dl-3-n-butylphthalide (NBP) to the injured hemisphere, making a single-dose (15 mg/kg) intravenous injection of NBP-encapsulated MSCsome facilitate the recovery of motor functions in model mice by improving the damaged microenvironment and suppressing neuroinflammation. This study underscores that the modification of the MSC membrane notably enhances the capacity of liposomes for precisely targeting the injured hemisphere, which is particularly crucial in treating cerebral ischemia-reperfusion injury.

DKK1 Activates the PI3K/AKT Pathway via CKAP4 to Balance the Inhibitory Effect on Wnt/β-Catenin Signaling and Regulates Wnt3a-Induced MSC Migration.

Wnt/β-catenin signaling plays a crucial role in the migration of mesenchymal stem cells (MSCs). However, our study has revealed an intriguing phenomenon where Dickkopf-1 (DKK1), an inhibitor of Wnt/β-catenin signaling, promotes MSC migration at certain concentrations ranging from 25 to 100 ng/mL while inhibiting Wnt3a-induced MSC migration at a higher concentration (400 ng/mL). Interestingly, DKK1 consistently inhibited Wnt3a-induced phosphorylation of LRP6 at all concentrations. We further identified cytoskeleton-associated protein 4 (CKAP4), another DKK1 receptor, to be localized on the cell membrane of MSCs. Overexpressing the CRD2 deletion mutant of DKK1 (ΔCRD2), which selectively binds to CKAP4, promoted the accumulation of active β-catenin (ABC), the phosphorylation of AKT (Ser473) and the migration of MSCs, suggesting that DKK1 may activate Wnt/β-catenin signaling via the CKAP4/PI3K/AKT cascade. We also investigated the effect of the CKAP4 intracellular domain mutant (CKAP4-P/A) that failed to activate the PI3K/AKT pathway and found that CKAP4-P/A suppressed DKK1 (100 ng/mL)-induced AKT activation, ABC accumulation, and MSC migration. Moreover, CKAP4-P/A significantly weakened the inhibitory effects of DKK1 (400 ng/mL) on Wnt3a-induced MSC migration and Wnt/β-catenin signaling. Based on these findings, we propose that DKK1 may activate the PI3K/AKT pathway via CKAP4 to balance the inhibitory effect on Wnt/β-catenin signaling and thus regulate Wnt3a-induced migration of MSCs. Our study reveals a previously unrecognized role of DKK1 in regulating MSC migration, highlighting the importance of CKAP4 and PI3K/AKT pathways in this process.

Intravitreal application of stem cells and their products does not ameliorate experimental autoimmune uveitis

Aims/Purpose: Stem cell therapy has demonstrated potential therapeutic benefits in various medical disciplines. This study evaluated therapeutic effects of mesenchymal stem cells (MSCs) or their by‐products on intraocular inflammation in experimental autoimmune uveitis (EAU) model.Methods: EAU was induced in C57BL/6J mouse strain through administration of IRBP, Freund's adjuvant and pertussis toxin. Conditioned medium was procured through cultivation of MSCs in serum‐free medium, while MSC supernatant was obtained via MSC centrifugation and membrane removal. Either conditioned medium or MSC supernatant was intravitreally administered unilaterally, typically on day 14 post‐EAU induction. Ocular fundus of mice was documented regularly. Experiments were generally concluded on day 28, with eyes processed for FACS and PCR analysis. Infiltration of retina by inflammatory cells (F4/80, CD11c, CD45, CD80, CD86, CD206) and gene expression of pro‐inflammatory factors (Gfap, Iba‐1, IL‐1, iNOS) were quantified.Results: Across all experiments, clinical grading, FACS, and PCR analysis indicated exacerbation of intraocular inflammation. Post‐intravitreal administration of MSC by‐products into eyes with EAU, increased infiltration of retina by inflammatory cells and upregulation in gene expression of pro‐inflammatory factors were observed. These findings were corroborated in vitro, where co‐culturing of retina with EAU in presence of MSCs or their supernatant did not significantly reduce gene expression of pro‐inflammatory factors. Upsurge in pro‐inflammatory factors was also noted in control experiments on healthy eyes post‐intravitreal injection of no substance, serum‐free medium, PBS, or conditioned medium.Conclusions: No improvement in EAU was observed following application of MSC products, both in vivo and in vitro. Instead, inflammation was more severe. Trauma induced by intravitreal application itself significantly influenced outcome.Financial support: GA UK 309621, SVV UK 260631.

A Nanodisc-Paved Biobridge Facilitates Stem Cell Membrane Fusogenicity for Intracerebral Shuttling and Bystander Effects.

Mesenchymal stem cell (MSC) therapies experience steadfast clinical advances but are still hindered by inefficient site-specific migration. An adaptable MSC membrane fusogenicity technology is conceptualized for lipid raft-mediated targeting ligand embedding by using toolkits of discoidal high-density lipoprotein (HDL)-containing biomimicking 4F peptides. According to the pathological clues of brain diseases, the vascular cell adhesion molecule 1 specialized VBP peptide is fused with 4F to assemble 4F-VBP (HDL), which acts as a biobridge and transfers VBP onto the living cell membrane via lipid rafts for surface engineering of MSCs in suspension. When compared with the membrane-modifying strategies of PEGylated phospholipids, 4F-VBP (HDL) provides a 3.86 higher linkage efficiency to obtain MSCs4F-VBP(HDL) , which can recognize and adhere to the inflammatory endothelium for efficient blood-brain barrier crossing and brain accumulation. In APPswe/PSEN1dE9 mice with Alzheimer's disease (AD), the transcriptomic analysis reveals that systemic administration of MSCs4F-VBP(HDL) can activate pathways associated with neuronal activity and diminish neuroinflammation for rewiring AD brains. This customizable HDL-mediated membrane fusogenicity platform primes MSC inflammatory brain delivery, which can be expanded to other disease treatments by simply fusing 4F with relevant ligands for living cell engineering.

Liposome-anchored mesenchymal stem cells for radiation pneumonia/fibrosis treatment

The effectiveness of mesenchymal stem cells (MSCs) on inflammation-related disease is limited and the pharmaceutical preparation that was used to enhance the efficacy of MSCs cannot reach the diseased tissue in large quantities. Herein, antioxidant liposome (Lipo-OPC) is designed to anchor onto the surface of MSCs membrane via click chemical reaction (MSC-Lipo-OPC) without affecting the viability and physiological characteristics of MSCs, thus allowing efficient accumulation of MSC-Lipo-OPC in X-ray irradiated lung sites. More importantly, MSC-Lipo-OPC promotes the change of the quantity and polarity of innate immunocytes, mainly including neutrophils, macrophages and Tregs, in favor of anti-inflammatory, finally preventing the formation of radioactive pulmonary fibrosis. Therefore, it could enhance the treatment outcome of both of MSCs and drugs to radiation-induced lung injury via modifying the drug-loaded nanoparticle on the surface of MSCs membrane, further promoting the application of MSCs in radiation damage and protection.

Stromal cell-derived factor-1α regulates chondrogenic differentiation via activation of the Wnt/β-catenin pathway in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been applied to treat degenerative articular diseases, and stromal cell-derived factor-1α (SDF-1α) may enhance their therapeutic efficacy. However, the regulatory effects of SDF-1α on cartilage differentiation remain largely unknown. Identifying the specific regulatory effects of SDF-1α on MSCs will provide a useful target for the treatment of degenerative articular diseases.To explore the role and mechanism of SDF-1α in cartilage differentiation of MSCs and primary chondrocytes.The expression level of C-X-C chemokine receptor 4 (CXCR4) in MSCs was assessed by immunofluorescence. MSCs treated with SDF-1α were stained for alkaline phosphatase (ALP) and with Alcian blue to observe differentiation. Western blot analysis was used to examine the expression of SRY-box transcription factor 9, aggrecan, collagen II, runt-related transcription factor 2, collagen X, and matrix metalloproteinase (MMP)13 in untreated MSCs, of aggrecan, collagen II, collagen X, and MMP13 in SDF-1α-treated primary chondrocytes, of glycogen synthase kinase 3β (GSK3β) p-GSK3β and β-catenin expression in SDF-1α-treated MSCs, and of aggrecan, collagen X, and MMP13 in SDF-1α-treated MSCs in the presence or absence of ICG-001 (SDF-1α inhibitor).Immunofluorescence showed CXCR4 expression in the membranes of MSCs. ALP stain was intensified in MSCs treated with SDF-1α for 14 d. The SDF-1α treatment promoted expression of collagen X and MMP13 during cartilage differentiation, whereas it had no effect on the expression of collagen II or aggrecan nor on the formation of cartilage matrix in MSCs. Further, those SDF-1α-mediated effects on MSCs were validated in primary chondrocytes. SDF-1α promoted the expression of p-GSK3β and β-catenin in MSCs. And, finally, inhibition of this pathway by ICG-001 (5 µmol/L) neutralized the SDF-1α-mediated up-regulation of collagen X and MMP13 expression in MSCs.SDF-1α may promote hypertrophic cartilage differentiation in MSCs by activating the Wnt/β-catenin pathway. These findings provide further evidence for the use of MSCs and SDF-1α in the treatment of cartilage degeneration and osteoarthritis.

Surface engineering of hollow gold nanoparticle with mesenchymal stem cell membrane and MUC-1 aptamer for targeted theranostic application against metastatic breast cancer

Mesenchymal stem cell membrane (MSCM)-coated biomimetic doxorubicin-loaded hollow gold nanoparticles were fabricated and decorated with MUC1 aptamer in order to provide smart theranostic platform. The prepared targeted nanoscale biomimetic platform was extensively characterized and evaluated in terms of selective delivery of DOX and CT-scan imaging.The fabricated system illustrated spherical morphology with 118 nm in diameter. Doxorubicin was loaded into the hollow gold nanoparticles through physical absorption technique with encapsulation efficiency and loading content of 77%±10 and 31%±4, respectively. The in vitro release profile demonstrated that the designed platform could respond to acidic environment, pH 5.5 and release 50% of the encapsulated doxorubicin during 48 h, while 14% of the encapsulated doxorubicin was released in physiological condition, pH 7.4 up to 48 h. The in vitro cytotoxicity experiments on 4T1 as MUC1 positive cell line illustrated that the targeted formulation could significantly increase mortality at 0.468 and 0.23 µg/ml of equivalent DOX concentration compared to non-targeted formulation while this cytotoxicity was not observed in CHO as MUC1 negative cell line. Furthermore, in vivo experiments showed high tumor accumulation of the targeted formulation even 24 h after intravenous injection which induced effective tumor growth suppression against 4T1 tumor bearing mice. On the other hand, existence of hollow gold in this platform provided CT scan imaging capability of the tumor tissue in 4T1 tumor bearing mice up to 24 h post-administration. The obtained results indicated that the designed paradigm are promising and safe theranostic system for fighting against metastatic breast cancer.

Engineered stem cell biomimetic liposomes carrying levamisole for macrophage immunity reconstruction in leukemia therapy

Most cancers recur after clinical treatment. Activation of the patient's own immunity can not only play a therapy role, but also consolidate the treatment prognosis. However, an effective immunity reconstruction strategy remains an impeding issue to be solved. Here, we report a macrophage immunity reconstruction strategy by engineering the stem cell biomimetic liposomes that carry levamisole (sLipo leva) to treat leukemia. The results demonstrated that sLipo leva was successfully constructed by incorporating the membrane of mesenchymal stem cells (MSCs). sLipo leva displayed a significant targeting effect on macrophages, induced the differentiation of them into M1 phenotype, exhibited a robust anticancer efficacy and an acceptable safety in leukemia-bearing mice. The immunity reconstruction mechanism of sLipo leva could be explained by two aspects: activation of macrophages themselves, and further activation of T cells, both of which contributed to the killing of leukemia cells. In conclusion, the present study offers a promising new strategy to activate the patient's own immunity for playing therapy efficacy and for consolidating treatment prognosis of leukemia.

Development of chimeric forms of the matrix metalloproteinase 2 collagen binding domain as artificial membrane binding proteins for targeting stem cells to cartilage lesions in osteoarthritic joints

Targeting stem cells to cartilage lesions has the potential to enhance engraftment and chondrogenesis. Denatured type II collagen fibrils (gelatin) are exposed in lesions at the surface of osteoarthritic articular cartilage and are therefore ideal target sites. We have designed and investigated chimeric mutants of the three modules of the MMP-2 collagen binding domain (CBD) as potential ligands for stem cell targeting. We expressed full-length CBD for the first time and used it to identify the most important amino acid residues for binding to gelatin. Module 2 of CBD had the highest affinity binding to both Type I and Type II gelatin, whereas module 1 showed specificity for type II gelatin and module 3 for type I gelatin. We went on to generate chimeric forms of CBD consisting of three repeats of module 1 (111), module 2 (222) or module 3 (333). 111 lacked solubility and could not be further characterised. However 222 was found to bind to type II gelatin 14 times better than CBD, suggesting it would be optimal for attachment to cartilage lesions, whilst 333 was found to bind to type I gelatin 12 times better than CBD, suggesting it would be optimal for attachment to lesions in type I collagen-rich tissues. We coated 222 onto the external membrane of Mesenchymal Stem Cells and demonstrated higher attachment of the coated cells to type II gelatin than uncoated cells. We conclude that the three modules of CBD each have specific biological properties that can be exploited for targeting stem cells to cartilage lesions and other pathological sites.

Irisin mediates beiging of adipose-derived mesenchymal stem cells through binding to TRPC3

BackgroundBeiging of white fat plays an important role in energy metabolism. Beige adipocytes contribute to the regulation of body weight and body temperature through expenditure of chemical energy to produce heat, and they have therefore recently attracted considerable attention as potential targets for therapeutic approaches in metabolic disorders, including obesity. All adipocytes, including beige adipocytes, differentiate from mesenchymal stem cells (MSCs), which may provide an important path for clinical intervention; however, the mechanism of beiging of human adipose cell-derived MSCs is not fully understood. Here, we provide insights on the role of IRISIN, which is known to be secreted by skeletal muscle and promote beiging of white fat.ResultsWe established an IRISIN-induced mesenchymal stem cell beiging model and found that IRISIN protein interacts with the MSC membrane protein TRPC3. This interaction results in calcium influx and consequential activation of Erk and Akt signaling pathways, which causes phosphorylation of PPARγ. The phosphorylated PPARγ enters the nucleus and binds the UCP1 promoter region. Furthermore, the role of TRPC3 in the beiging of MSCs was largely abolished in Trpc3−/− mice. We additionally demonstrate that the calcium concentration in the brain of mice increases upon IRISIN stimulation, followed by an increase in the content of excitatory amino acids and norepinephrine, while Trpc3−/− mice exhibit the reverse effect.ConclusionsWe found that TRPC3 is a key factor in irisin-induced beiging of MSCs, which may provide a new target pathway in addressing metabolic disorders. Our results additionally suggest that the interaction of irisin with TRPC3 may affect multiple tissues, including the brain.Graphical abstract

Constructions of ROS-responsive titanium-hydroxyapatite implant for mesenchymal stem cell recruitment in peri-implant space and bone formation in osteoporosis microenvironment.

To solve the issue of unsatisfactory recruitment of mesenchymal stem cells (MSCs) around implant in osteoporotic fractures, we fabricated a ROS-responsive system on titanium surface through hydroxyapatite coating and biomolecule grafting. The porous hydroxyapatite and phosphorylated osteogenic growth peptides (p-OGP) were introduced onto titanium surface to synergistically improve osteogenic differentiation of MSCs. After the p-OGP-promoted expression of osteogenic related proteins, the calcium and phosphate ions were released through the degradation of hydroxyapatite and integrated into bone tissues to boost the mineralization of bone matrix. The ROS-triggered release of DNA aptamer (Apt) 19S in the osteoporotic microenvironment guides MSC migration to implant site due to its high affinity with alkaline phosphatase on the membrane of MSCs. Once MSCs reached the implant interface, their osteogenic differentiation potential was enhanced by p-OGP and hydroxyapatite to promote bone regeneration. The study here provided a simple and novel strategy to prepare functional titanium implants for osteoporotic bone fracture repair.

Monocytic Myeloid-Derived Suppressor Cells Inhibit Myofibroblastic Differentiation in Mesenchymal Stem Cells Through IL-15 Secretion.

Background: Accumulating evidence indicates that mesenchymal stem cells (MSCs) are precursors of myofibroblasts, which play a vital role in renal fibrosis. The close interaction between MSCs and other immune cells regulates the development of multiple fibrosis-related diseases. However, the effect of myeloid-derived suppressor cells (MDSCs) on MSCs remains unexplored. Here, we investigated the effect of MDSCs on the myofibroblastic differentiation of MSCs. Methods: MSCs were induced to undergo myofibroblastic differentiation with transforming growth factor beta 1 (TGF-β1). M-MDSCs and G-MDSCs were sorted by flow cytometry. Supernatants derived from MDSCs were administered to cultured bone marrow MSCs (BM-MSCs) undergoing TGF-β1-induced myofibroblastic differentiation. Myofibroblastic differentiation was evaluated by immunostaining. The expression of fibrosis-related genes was determined by quantitative PCR and western blot analysis. In vitro, M-MDSC supernatant or M-MDSC supernatant with interleukin (IL)-15 mAbs was administered following unilateral renal ischemia-reperfusion injury (IRI) to observe the myofibroblast differentiation of renal resident MSCs (RRMSCs) in a murine model. Results: Myofibroblastic differentiation of MSCs was hindered when the cells were treated with MDSC-derived supernatants, especially that from M-MDSCs. The inhibitory effect of M-MDSC supernatant on the myofibroblastic differentiation of MSCs was partially mediated by IL-15-Ras-Erk1/2-Smad2/3 signaling. Treatment with M-MDSC supernatant ameliorated renal fibrosis and myofibroblastic differentiation in RRMSCs through IL-15. Additionally, M-MDSC supernatant increased M-MDSC infiltration in the kidney in a mouse IRI model. M-MDSC supernatant downregulated the adhesion and migration marker CD44 on the cell membrane of MSCs via IL-15. Conclusion: M-MDSC-derived supernatant inhibited the TGF-β1-induced myofibroblastic differentiation of MSCs through IL-15. Our findings shed new light on the effect of MDSCs on myofibroblastic differentiation and adhesion of MSCs, which might provide a new perspective in the development of treatment strategies for renal fibrosis.

Mesenchymal stem cell secretome and nanotechnology: Combining therapeutic strategies

Mesenchymal stem cells (MSC) have pushed the field of stem cell-based therapies by inducing tissue regeneration, immunosuppression, and angiogenesis mainly through vesicles and soluble factors release (paracrine signaling). MSC-extracellular vesicles (MSC-EV) adaptable secretome and homing to injured sites allowed researchers to unlock a new era of cell-free based therapy. In parallel, nanoparticles (NP) have been explored in contributing to transport and drug delivery systems, giving drugs desired physical-chemical properties to exploit cell behavior. However, NPs can be quickly recognized by immune cells and cleared from circulation. In this viewpoint, we explore how combining both therapeutic strategies can improve efficacy and circumvent limitations of both therapies. MSCEV benefit from the potent MSC membrane composition, guiding chemotaxis to tumor sites, a very restricted microenvironment. MSC-EV has low immunogenicity, high stability, long half-life and can explore tissue targeting ligands as a precise drug carry, even across biological barriers. Those properties promote enhanced targeted drug delivery that can be combined with NP, exploring biological membrane production through: 1. direct cell therapy with NP-infused MSC; 2. NP-containing MSC-EV generated by NP-infused MSC; 3. by coating NP in MSC membrane (“MSC NanoGhosts”), allowing precise cargo definition without losing targeting. Therefore, nanotechnology combined with cell-based therapeutic resources can greatly improve targeted drug delivery, improving efficacy and opening a new venue of therapeutic possibilities.

Co-modification with MSC membrane and PDA prevents Fe3O4-induced pulmonary toxicity in mice via AMPK-ULK1 axis

Fe3O4 nanoparticles are widely used in the diagnosis and treatment of diseases due to their superparamagnetism, but their toxicity in vivo, which can result in apoptosis or autophagy, cannot be ignored. It has been reported that polydopamine (PDA) modification can reduce the toxicity of Fe3O4 and increase its biocompatibility. However, more research is warranted to further improve the modification method. We therefore developed a new method to coat Fe3O4@PDA nanoparticles with the mesenchymal stem cell membrane (MSCM) and evaluated the toxicity of the modified particles in the lungs of mice.We found that the MSCM modification significantly reduced lung injury induced by Fe3O4 particles in mice. Compared with Fe3O4@PDA nanoparticles, co-modification with MSCM and PDA significantly reduced autophagy and apoptosis in mouse lung tissue, and reduced activation of autophagy mediated by the AMPK-ULK1 pathway axis. Thus, co-modification with MSCM and PDA prevents Fe3O4-induced pulmonary toxicity in mice by inhibiting autophagy, apoptosis, and oxidative stress.

Biomimetic Black Phosphorus Nanosheet-Based Drug Delivery System for Targeted Photothermal-Chemo Cancer Therapy

As a biodegradable material, black phosphorus (BP) has been considered as an efficient agent for cancer photothermal therapy. However, its systemic delivery faces several hurdles, including rapid degradation in blood circulation, quick clearance by the immune system, and low delivery sufficiency to the tumor site. Here, we developed a biomimetic nanoparticle platform for in vivo tumor-targeted delivery of BP nanosheets (BP NSs). Through a biomimetic strategy, BP NSs were utilized to coordinate with the active species of oxaliplatin (1,2-diaminocyclohexane) platinum (II) (DACHPt) complexions, and the nanoparticles were further camouflaged with mesenchymal stem cell (MSC)–derived membranes. We showed that the incorporation of DACHPt not only decelerated the BP degradation but also enhanced the antitumor effect by combining the photothermal effect with chemotoxicity. Furthermore, MSC membrane coating increased the stability, dispersibility, and tumor-targeting properties of BP/DACHPt, significantly improving the antitumor efficacy. In short, our work not only provided a new strategy for in vivo tumor-targeted delivery of BP NSs but also obtained an enhanced antitumor effect by combining photothermal therapy with chemotherapy.

Active stealth and self-positioning biomimetic vehicles achieved effective antitumor therapy

Mesenchymal stem cells (MSCs) are recognized as promising drug delivery vehicles. However, the limitation of drug loading capacity and safety considerations are two obstacles to the further application of MSCs. Here, we report MSC membrane-coated mesoporous silica nanoparticles (MSN@M) that maintain the active stealth and self-positioning drug delivery abilities of MSCs and resolve issues related to MSCs-mediated drug delivery. MSN@M was established through uniformly integrating MSC membrane onto a mesoporous silica nanoparticle (MSN) core by sonication. Reduced clearance of phagocytes mediated by CD47 marker on MSC membrane was observed in vitro, which explained the only ~ 25% clearance rate of MSN@M compared with MSN in vivo within 24 h. MSN@M also showed stronger tumor targeting and penetration ability compared with MSN in HepG2 tumor bearing mice. Simultaneously, MSN@M exhibited strong capacity for drug loading and sustained drug release ability of MSN when loaded with doxorubicin (DOX), the drug loading of MSN@M increased ~ 5 folds compared with MSC membrane. In HepG2 xenograft mice, DOX-loaded MSN@M effectively inhibited the growth of tumors and decreased the side effects of treatment by decreasing the exposure of other tissues to DOX. Consequently, our MSN@M may serve as alternative vehicles for MSCs and provide more options for antitumor treatment.

Presence of Clustered GM1 Ganglioside in the Membrane of Endometrial Mesenchymal Stem Cells is Dependent on Cell Cycle Stage

Gangliosides, representing the group of sialic glycosphingolipids, are typical components of lipid rafts of cell membranes and play an important role in the processes of reception and signal transduction. Gangliosides attract specific attention as possible regulators of directed differentiation of mesenchymal stem cells (MSCs). GM1 ganglioside is considered as a marker of lipid microdomains, and fluorescence labeling of clusters of GM1 molecules is one of the main methods for assessing the integrity or destruction of rafts in various types of cells. At the same time, data on the presence and possibility of determining GM1 ganglioside in the plasma membrane of MSCs are extremely limited. The objective of this work was to identify the ganglioside GM1 in the membrane of human endometrial MSC (eMSC) using an experimental approach based on interaction with the pentamer of the beta subunits of cholera toxin (CTB). Fluorescence staining of cells using FITC-CTB conjugate revealed the heterogeneity of the eMSC culture by the presence of GM1 membrane clusters. Experiments on synchronized culture have shown that the amount of clustered GM1 in the eMSC plasma membrane depends on the phase of the cell cycle: it is maximum when the cells are stopped at the G0/G1 phase boundary and decreases when proliferation starts, reaching a minimum in the G2/M phases. The data suggest the functional role of GM1-containing lipid rafts in cell cycle of eMSCs and possible changes in the mechanical properties of the membrane before cell division.

Endoglin Expression and Surface Renewal in Mesenchymal Stem Cells and Endothelial Cells

Endoglin (CD105) is one of the major marker antigens of mesenchymal stem cells (MSC) and endothelial cells. While the functions of endoglin in endothelial cells have been widely declared, little is known about its role in MSC biology. Here, we present a comparative study of CD105 expression, interna-lization and shedding by human endothelial cell line EA.hy926 and human adipose tissue-derived MSC from various sources. While the fraction of CD105-positive cells in all MSC cultures and EA.hy926 endothelial cells was consistently high, over 97% of the population, the density of CD105 molecules on the surface of MSC isolated from visceral and subcutaneous adipose tissue was substantially different. The total expression level of endoglin mRNA in MSC and endothelial cells was similar, whereas the contribution of the transcripts that yield a short CD105 isoform was higher in endothelial cells. Using a set of monoclonal antibodies (mAbs) directed against different endoglin epitopes, we revealed significant differences in the dynamics of CD105 metabolism on the membranes of endothelial cells and MSC. On EA.hy926 endothelial cells, CD105 molecules bound with antibodies were internalized and remained in the perinuclear space. In MSC cultures, on the contrary, the CD105-mAbs complexes were not subjected to endocytosis and remained on the cell membrane for a long time. We demonstrated that MSC, similarly to the endothelial cells, do shed the extracellular endoglin fragment into the environment as a soluble CD105 isoform. The shedding process in MSC was, however, substantially less intensive compared with the endothelial cells. Thus, we revealed, for the first time, that in MSC, unlike the endothelial cells, endoglin persists on cell surface for a long time and is not interna-lized after binding with antibodies. Endoglin shedding from MSC surface and the concomitant generation of the soluble endoglin form were also demonstrated for the first time.

TGase-mediated cell membrane modification and targeted cell delivery to inflammatory endothelium

Targeted cell delivery to lesion sites via minimally invasive approach remains an unmet need in regenerative medicine to endow controlled cell distribution and minimized side-effects. Current cell modification approaches to improve cell delivery tend to have adverse effects on cellular phenotype and functionality. Here, we rationally developed a facile and mild cell modification and targeted delivery strategy leveraging endogenous tissue transglutaminase (TGase) expressed on the surface of MSCs (Mesenchymal Stem Cells) and inflammatory endothelial cells (ECs). Cell modification by functional peptides was accomplished simply via TGase catalyzed cross-linking with naturally-expressed MSCs membrane proteins (e.g. Annexin II), without detectable disturbance of cellular viability and functionality. The modified functional peptides could facilitate adhesion of MSCs to inflammatory ECs (with up-regulated TGase expression compared with normal ECs) in vitro, as demonstrated by a one-fold increase of the MSC-EC adhesion force measured by atomic force microscopy (AFM) and by targeted delivery of modified MSC to inflammatory ECs in a flow chamber assay. When transplanted in vivo, modified MSCs demonstrated a dramatic increase in targeted efficiency to inflammatory endothelium compared with non-modified MSCs in both mice ear inflammation and acute/chronic liver injury models. The cell membrane modification strategy and targeted cell delivery mechanism described here can be readily extended for empowering cell engineering and cell therapy with multifaceted functionalities to combat refractory diseases.