Mesenchymal Stem Cell Engineered Nanovesicles for Accelerated

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Tissue Engineering and Regenerative Medicine

Mesenchymal Stem Cell Engineered Nanovesicles for Accelerated Skin Wound Closure Chungmin Han, Dayeong Jeong, Bumju Kim, Wonju Jo, Hyejin Kang, Siwoo Cho, Ki Hean Kim, and Jaesung Park ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01646 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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ACS Biomaterials Science & Engineering

Mesenchymal Stem Cell Engineered Nanovesicles for Accelerated Skin Wound Closure Chungmin Hana, Dayeong Jeongb, Bumju Kimc, Wonju Joa, Hyejin Kangb, Siwoo Choa, Ki Hean Kimac and Jaesung Parkab* a Department

of Mechanical Engineering, POSTECH, 77 Cheongam-Ro, Nam-Gu, Pohang,

Gyeongbuk, 37673, Republic of Korea b

School of Interdisciplinary Bioscience and Bioengineering, POSTECH, 77 Cheongam-Ro,

Nam-Gu, Pohang, Gyeongbuk, 37673, Republic of Korea c

Division of Integrative Biosciences and Biotechnology, POSTECH, 77 Cheongam-Ro, Nam-

Gu, Pohang, Gyeongbuk, 37673, Republic of Korea *

Correspondence: [email protected]

KEYWORDS. Extracellular vesicles, Nanovesicles, Mesenchymal Stem cells, Skin regeneration

ABSTRACT We report development and characterization of cell-engineered nanovesicles made from mesenchymal stem cells (MSCNVs), which have more than 300 times higher productivity than natural extracellular vesicles (EVs). MSCNVs had similar morphological characteristics to MSCEVs, but have molecular characteristics that more resemble MSCs than MSCEVs. In vitro MSCNV treatment increased the proliferation and migration of primary skin fibroblasts, and

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showed better effects than treatment using natural MSCEVs. Quantitative real-time PCR analysis showed increased expression of growth factors in MSCNV-treated skin fibroblasts. Intraperitoneal injection of MSCNVs into syngeneic mice induced mild local inflammation, which resulted in recruitment of immune cells to the injection site. In vivo MSCNV treatment of a mouse skin wound accelerated its healing; this acceleration by MSCNVs may occur by promoting blood-vessel formation at the wound site. These results indicate the promise of MSCNVs as agents for regenerative medicine.

Introduction Mesenchymal stem cells (MSCs) are adult stem cells that function in various physiological activities1–3. MSCs can stimulate tissue regeneration, and therefore may have applications in regenerative medicine 4,5. Tissue regeneration by MSCs is affected by both direct differentiation of MSCs at the injection site and by soluble growth factors and extracellular matrices (ECMs) that MSCs secrete

6,7.

However, direct cell implantation for therapy raises safety concerns because

proliferative cells can move away from the injection site, change their behavior and multiply in number7,8. Therefore, approaches that use purified or concentrated MSC by-products have been developed 9,10. Extracellular vesicles (EVs) secreted by MSCs can also accelerate regeneration of damaged tissues

11–13.

Therapeutic approaches that use MSCEVs do not have the side effects of

direct cell implantations 8. Use of EVs in therapy is impeded by their low availability in in vitro culture conditions. A typical yield of EVs from cultured MSCs is < 1 mg/L of cell-cultured medium, which is too low for harvest to be practical for therapeutic use in humans

14.

MSCs also have finite lifespans, so this yield

problem is especially critical: purifying even a single dosage of active MSCEV for an adult human


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is challenging. To overcome this yield limitation, cell-engineered nanovesicles (CNVs) have been developed as an alternative to EVs. CNVs are mechanically manufactured by repeatedly passing cells through a microporous filter; this process shreds the cells’ membranes into fragments, wihch spontaneously reassemble into nanometer-size vesicles 15. CNVs contain cellular materials such as RNAs and proteins from the cells that were used to produce them 16. Due to these characteristics, CNVs can convey the cells’ function in a way that is similar to the functions of EVs. CNVs produced from mouse embryonic stem cell successfully increase the proliferation of skin fibroblasts and MSCs in vitro 17. In this research, we generated CNVs from MSCs (MSCNVs) to treat skin wounds. First, we compared the effects of MSCNVs with the effects of EVs generated from MSCs (MSCEVs) in treatment of syngeneic primary skin fibroblast. Skin fibroblasts treated with MSCNVs showed increased cell proliferation and migration, and activated signaling pathways that are related to cell proliferation. Application of MSCNV to skin fibroblasts induced increased expression of two angiogenic growth factors: TGF-β and VEGF-α. When MSCNVs were injected into syngeneic mice, they seemed to induce mild local inflammation. To confirm that MSCNVs accelerate healing of skin wounds, a mouse-skin wound model was treated with MSCNVs and wound-healing effects were evaluated by naked-eye observation and optical coherence tomography (OCT). Wounds treated with MSCNVs regenerated faster than non-treated wounds.

Experimental (Materials and Methods) Mouse primary skin fibroblasts and MSC preparation.


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Skin fibroblasts were isolated from C57BL/6 mice by following a protocol that was slightly 18.

modified from a previous study

Fibroblasts were then cultured in DMEM (Hyclone)

supplemented with 10% fetal bovine serum (FBS, Hyclone) and 100 μg/mL penicillinstreptomycin (Gibco). Cells from passage numbers six to eight were used for in vitro studies, because these cells have a low proliferation rate. For proliferation studies, skin fibroblasts were serum-starved with DMEM 1% FBS for 24 h to synchronize their cell cycles to the G1 phase, then treated with MSCNVs or MSCEVs. MSCs were isolated from 8-week-old male C57BL/6 mice. The mice were sacrificed by cervical dislocation and their femurs and tibiae were carefully separated. Attached muscle and connective tissues were removed from each bone, then the bones were soaked in phosphate-buffered saline (PBS). Both ends of bones were slightly cut with scissors, then bone marrow was extracted by injecting PBS through a 26-gauge needle repeatedly until the bone became transparent. Collected bone marrow solution was filtered through a cell strainer (BD, 70 μm), then the filtered solution was centrifuged at 4°C for 5 min at 200 x g. Supernatant was removed, then the pellet of bone marrow cells was resuspended in MEM-α containing 15% FBS and the cells were seeded on 100mm cell culture dish. After plating the cells on the culture dish, cells were cultured for 4 d without medium change to facilitate cell adhesion to the dish. On the fourth day, attachment of the cells was confirmed, then the medium was carefully changed to MEM-α containing 10% FBS. MSCs were sub-cultured once and a portion of the cells was used for confirmation of MSC-related surface markers. MSCs that were expressing appropriate surface markers (passage number 1) were cryopreserved in liquid nitrogen until they were used in experiments. All MSCs used in this study were from the same isolation batch. MSC characterization by flow cytometry


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To confirm that isolated MSCs had been extracted properly and had specific characteristics, we conducted flow cytometry. The types of cells can be identified by the presence or absence of specific cell surface markers. MSC is positive protein CD44, and negative for proteins CD11b and CD45. Culture MSCs were detached and suspended in PBS containing 2% FBS; cell population was 2.0 × 107 cells/mL. Antibodies of CD44 (BD biosciences, 553133), CD11b (BioLegend, 101211) and CD 45 (BioLegend, 103125) were added to the cell suspension, and it was stored on ice for 30 min. The cell solution was washed by centrifugation at 840xg for 5 min. The supernatant was discarded and the pellet was resuspended with PBS containing 2% FBS and then the solution was also centrifuged at 840 x g for 5 min. The cell solution was poured into test tube and analyzed using LSRFortessa (BD biosciences). MSCNV preparation The MSCs from passage numbers five to eight were used for CNV preparations. MSCs were cultured in 150-mm tissue culture dishes until they reach > 90% confluence (~3 days for MSCs thawed from liquid nitrogen; ~2 days for normal MSCs). To preserve the surface proteins, the cells were then detached using 2 mM Ethylenediaminetetraacetate (EDTA)/PBS instead of using enzymatic reagents such as trypsin for preserving their surface proteins. The cells were pelleted and resuspended at 1.0×108 cells/mL in PBS. The cell suspension was repeatedly passed through a polycarbonate filter which was installed in a mini-extruder (Avanti). Cells were first passed through 10-μm pore-size polycarbonate membranes (Whatman) five times and then passed through 5-μm pore-size membranes (Whatman) five times in a newly-assembled device. To remove unbroken cells and large cell debris, the resulting extrusion solution was centrifuged at 1000 x g for 5 min. MSCNVs in the extrusion solution were separated by iodixanol density-gradient centrifugation. Then Opti-prep (AXIS-SHIELD) layers were prepared from 10% Opti-prep


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solution on top of 30% Opti-prep solution in an ultra-centrifuge tube. The pre-cleaned cell extrusion solution was layered on top of the 10% Opti-prep, then ultra-centrifuged at 1 × 105 x g for 1 h. The MSCNVs were isolated between the 10% and 30% Opti-prep layers. The MSCNVs were carefully collected and quantified using a Bradford protein assay. MSCEV preparation MSCs from passage numbers five to eight were used for EV preparations. Depleted FBS was prepared by ultracentrifugation of FBS at 100,000 x g for 16 h. The pellet containing serumderived vesicles was discarded, and the vesicle-free supernatant was used for the MSC culture. When MSC cultures had grown to ~90 % confluence, culture medium was changed to MEM-α supplemented with 10% EV-depleted FBS. After incubation for 24 - 48 h, the cultured medium was collected and centrifuged at 500 x g for 10 min to remove dead cells and large cell debris. The supernatant was centrifuged again at 3,000 x g for 20 min to remove small cell debris, then ultracentrifuged at 1 × 105 x g for 2 h. The supernatant was discarded and the pellet containing MSCEVs was resuspended in PBS. The MSCEV solution was ultra-centrifuged again at 1×105 x g for 2 h to remove residual protein contaminants. Finally, the supernatant was discarded and the pellet was suspended in 100 μL PBS and quantified using a Bradford protein assay. Western blotting The samples were lysed using RIPA buffer (Bio-Rad) and quantified using BCA protein assay. Samples were then loaded into appropriate polyacrylamide gels and electrophoresed. The separated protein bands in the gel were transferred onto Polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in 0.1% tween 20 in Tris-buffered saline (TBS) at RT for 1 h. The primary antibodies were diluted to 1:1000 with blocking solution


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and incubated with the sample at 4 °C overnight. PVDF membranes were washed with 0.05% tween 20 in TBS (TBST), then secondary antibodies were diluted to 1:5000 with blocking solution and incubated at RT for 1 h. The membranes were washed with TBST and reacted with a chemiluminescent substrate (Thermo scientific). The signals were imaged using c-Digit scanner (LI-COR). The primary and secondary antibodies used in the experiment were as follows: Antiactin (sc-81178,) Anti-calnexin (SC-11397), Anti-CD29 (integrin beta 1, sc-374429), Anti-CD9 (sc-51575), Anti-CD81 (sc-166029) and Anti-cMyc (sc-788) were purchased from Santa Cruz Biotechnology Inc., Anti-PCNA (ab2426) and Anti-CD105 (ab21222) were purchased from Abcam, Anti-MAPK (#9107), Anti-p-MAPK (#4370), Anti-STAT3 (#9139) and Anti-p-STAT3 (#9145) were purchased from Cell signaling Technology and Anti-mouse HRP (sc-2005), Antigoat HRP (sc-2020) and Anti-rabbit HRP (sc-2004) were purchased from Santa cruz. RNA profile analysis RNAs in the MSCNVs, MSCEVs and MSCs were isolated using Tri reagent (Sigma) and chloroform (Sigma). RNA concentration was measured using a NanoVue device (GE Healthcare) and the RNA profiles were analyzed using an Agilent RNA 6000 Nano Chip with a 2100 Bioanalyzer (Agilent Technologies). Nanoparticle tracking analysis MSCNVs and MSCEVs were diluted with particle-free PBS that had been filtered through a 0.22μm filter. The numbers and sizes of CNVs were measured using Nanosight LM10 (Malvern Instruments). The concentrations were adjusted until images contained < 100 particles. All samples were measured at least three times. Average particle counts and standard deviations were calculated from three measurements.


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Transmission electron microscopy A transmission electron microscope (TEM, Joel 10011, Japan) was used to confirm the size and shape of MSCNVs and MSCEVs. Samples were loaded onto formavar carbon film (Electron Microscopy Science) for 5-7 s then negatively stained with 2% uranyl acetate for image contrasting. The grid was completely dried at room temperature (RT) before imaging. Reverse transcriptase (RT) polymerase chain reaction (PCR) RT-PCR analysis was performed to analyze mRNA contents of MSCNVs. mRNAs were isolated from MSCNVs, skin fibroblasts and MSCNV-treated skin fibroblasts by using Tri-reagent (Sigma) according to supplier’s manual. The isolated mRNAs were quantified using NanoVue (GE healthcare) and were transcribed to cDNAs by using an ImProm-II reverse transcription system and oligo dT primers (Promega). cDNAs were then applied to PCRs with primers that probe the sequences of β-actin, VEGF-α or TGF-β. Primers used for amplification were: β-actin (NM_007393.5,

fwd:

5’-ACGTTGACATCCGTAAAGAC-3’,

GCAGTAATCTCCTTCTGCAT-3’,

100

bp)

TGF-β

(NM_011577.2,

rev: fwd:

5’5’-

CTGCTGACCCCCACTGATAC-3’, rev: 5’-GCCCTGTATTCCGTCTCCTT-3’, 93 bp) VEGFα

(NM_001025250.3,

fwd:

5’-CACCCACGACAGAAGGAGAG-3’,

rev:

5’-

TCTCAATCGGACGGCAGTAG-3’, 87 bp). The PCR products were separated on a 1% agarose gel which was prepared with SYBR® Green (molecular probe). The gel images were taken using a BioDoc-It imaging system (UVP). Quantitative polymerase chain reaction (qPCR) To measure relative expression levels of VEGF-α and TGF-β, the cDNA from MSCNV-treated skin fibroblasts was also analyzed by real-time qPCR using an iQ SYBR Green Supermix kit (Bio-


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Rad) and MyiQ Real-time PCR Detection System (Bio-rad). Primers were the same as those used in the RT-PCR. Measured gene expressions of VEGF-α and TGF-β were normalized by β-actin expression and further compared by the normalized expression of untreated skin fibroblasts. In vitro proliferation test Skin fibroblasts were seeded on six-well plates at 30,000 cells/well and cultured for 24 h. The culture medium was then replaced with DMEM containing 1% FBS for 24 h. The cells were then treated with 50 μg/mL of MSCNVs or MSCEVs in DMEM containing 1% FBS for 48 h. The EVtreatment groups were used as controls in most of the experiments because the effects of the EVs have been demonstrated elsewhere. The dosages of CNVs and EVs were determined in previous CNV studies 17. After the treatment, the cells were washed once and trypsinized (Gibco) for cell counting using a haemocytometer under a phase-contrast microscope. All samples were prepared in triplicate and more than three sections of haemocytometer were counted for each counting. Average cell counts and standard deviations were calculated from triplicated samples. Scratch-closure assay A scratch closure assay was performed following the protocol published elsewhere19. Skin fibroblasts were seeded on 12-well plates and cultured to confluence. Some of the wells were treated with 15 μg/mL mitomycin-C (MMC) in complete growth medium for 2 h to arrest growth. Confluent cell layers were then incubated in DMEM containing 1% FBS for 24h. The cell layers were scratched in a straight line with a 200-μL micro-pipet tip. To remove detached cell and debris, the samples were washed with DMEM. DMEM containing 1 % FBS and 50 μg/mL of MSCNVs or MSCEVs was added to the scratched cell layers. Images were taken at a marked point of the scratched area every 24 h through a phase-contrast microscope. To confirm wound closure, the


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gap distances of scratches were measured using imageJ software (NIH). All samples were prepared in triplicate, and the images were taken from the same marked area for consistency of measurement. In vivo mouse immune response Seven-week-old female C57BL/6 mice were used to test the effect of MSCNVs on inflammatory response in vivo. Mice were separated into two groups; one group received an intraperitoneal injection of MSCNVs (50 μg in 100 μl PBS) and the other group received an intraperitoneal injection of same volume PBS. The mice were anesthetized by intramuscular injection (~240 mg/kg) of 2,2,2-tribromoethanol (Sigma) at 24 h after the injections. Peritoneal cavities were washed with 3 ml ice-cold PBS and total bloods were then collected by cardiac puncture from the anesthetized mice. Peritoneal wash solutions were centrifuged at 200 x g for 10 min, then resuspended in 5 mL PBS; cell numbers were counted using a haemocytometer. Sera were prepared from whole bloods which were allowed to clot for 30 min at RT. Blood clots were removed by centrifugation at 1,000 x g for 10 min and serum fractions (supernatant) were collected and analyzed using a mouse cytokine/chemokine ELISA kit (Multi-Analyte ELISArray Kits, QIAGEN) according to the supplier’s manual. In vivo mouse-wound model Seven-week-old female C57BL/6 mice were used in the wound-model experiment. On the operation day, mice were anesthetized by intramuscular injection (!240 mg/kg) of 2,2,2tribromoethanol (Sigma). Dorsal hairs were removed using a hair clipper, and the hair roots were removed using a depilatory cream (Ildong). Two round full-thickness wounds of 4-mm diameter


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were created on each side of the back of each mouse by using 4-mm diameter biopsy punch (Miltex). MSCNVs (500 μg/ml) were suspended in PBS, then injected using 31-gauge needle syringes. MSCNVs were subcutaneously injected to the healthy skin tissue bordering the wounds in multiple injection sites surrounding the wounds. A total of 100-μL MSCNV solution was injected to apply 50 μg MSCNVs to the wounds; the same volume of PBS was applied to the wound on the other side. To prevent skin contraction, O-ring-shaped silicone sheets were attached around the wound area by using medical adhesive spray (Hollister). A Transparent Tegaderm (3M Health Care) was applied to the silicone sheet to protect the wound from infections. Tegaderms did not interfere with OCT imaging, so they were not removed until the last day of the experiment. Animal behavior and dressing status were monitored every day during the experiment. Wounds were checked every 3 d after operation. Hematoxylin and eosin staining Skin tissues from 7th day mouse wounds were preserved in 4% paraformaldehyde for 24 h, then embedded in paraffin. Serial tissue sections of 4-μm thickness were prepared, stained with hematoxylin and eosin (H&E) and observed under a microscope (Olympus, DP 71). Optical coherence tomography To monitor the wounds by using OCT, mice were anesthetized by inhalation anaesthesia of 2% isoflurane and oxygen. Each wound area was imaged in 3D by using a custom OCT system 20. The imaging field of view (FOV) was 5mm x 5mm x 1.4mm (x, y, and z). The image resolution was 20µ in the lateral direction and 10.8µm in the axial direction. 3D OCT images consisting of 500 x 500 x 320 pixels (x, y, and z) were obtained. For angiographic OCT imaging, 10 cross-sectional images in the x-z direction were acquired at each y position in the sample and were processed


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using a complex differential variance method. The imaging time was approximately 50 s per volume. Statistical analysis All in vitro experiments were performed with triplicated samples. The results were displayed with mean values ± standard deviations. The results also statistically analyzed using Student’s t-tests, and those that were significantly different were marked: * (p < 0.05); ** (p < 0.01).

Results Production and characterization of MSCNVs Murine MSCs were isolated from B57BL/6 mouse; the MSC specific markers were checked using a flow cytometer. Isolated cells were negative for CD11b and CD45, but positive for CD44 (Figure S1. MSC characterization). MSCNVs were purified using a method developed previously 17. MSC EVs were isolated by conventional ultracentrifugation 21. Produced CNVs and isolated EVs were analyzed and compared (morphology, size, contents of protein and RNA). Only MSCs from passages five to eight were used for CNV and EV preparation because MSCs from high passage numbers (e.g. passage 11) tend to show decreased MSC marker expressions. NVs and EVs were similar in some ways, but different in others. CNVs were spherical with diameter ~100 nm, which is similar to that of MSC EVs (Figure 1 a). The sizes of NVs and EVs both ranged from ~ 100 nm to ~ 200 nm (Figure 1 b). The protein bands of MSCNVs were evenly distributed from 17 kDa to 225 kDa; those of MSCEVs mostly showed large proteins (Figure 1 c). The protein band profile of MSCNV proteins was similar to that of MSCs but not to that of


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MSCEVs (Figure 1 c). Western blot analyses detected Actin and Calnexin in cells and CNVs, but not in EVs. CD29 was observed in all cases, but CD105 was only observed in MSCs and MSCNVs (Figure 1 d). CD9 and CD81 were mostly observed in MSCEVs, whereas MSCs and MSCNVs showed almost no expression of the proteins (Figure 1 d). Actin is a housekeeping protein of cells and Calnexin is a marker for endoplasmic reticulum (ER). CD105 and CD29 are MSC-related markers and CD9 and CD81 are EV-related markers. Total RNA chip analyses showed almost the same RNA expression profiles in MSCs and MSCNVs, but MSCEVs did not show typical 5s, 18s or 28s RNA peaks (Figure 1 e). To further analyze the mRNA contents of samples, reverse transcriptase PCR analysis was performed using oligo dT primers. MSCEVs contain too few mRNAs to make cDNA for further analysis. Analysis of cDNAs from MSCs and MSCNVs by additional PCR using primers that probed for β-actin, VEGF-α and TGF-β showed almost no differences between the samples (Figure 1 f). Quantitative PCR analysis of gene expressions with the same primers also showed almost no differences between MSCs and MSCNVs (Figure S2. qPCR analysis of MSCs and MSCNVs).


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Figure 1 a. TEM image of MSCEVs and MSCNVs. MSCEVs and MSCNVs were ~50 nm to 200 nm in diameter and showed similar morphology under TEM observation. Bars: 200 nm. b. NTA analysis of MSCEVs and MSCNVs. MSCEVs and MSCNVs showed peak diameter at ~100 nm. Solid line: mean value. Grey area: standard deviation c. SDS-PAGE and Coomassie staining of


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MSCEVs and MSCNVs. MSCs and MSCNVs showed similar protein staining pattern over various sizes. MSCEV showed distinctively different protein staining pattern. d. Protein expressions of MSCEVs and MSCNVs. MSCNVs expressed cellular proteins (actin and calnexin) but MSCEVs did not. MSCNVs expressed two MSC-specific membrane proteins (CD105 and CD29) but MSCEV expressed only CD29. MSCEV expressed EV-specific markers (CD81 and CD9) but MSCNV did not. e. RNA profile of MSCEVs and MSCNVs. RNA expression profile of MSCNVs were almost the same as the RNA profile of MSCs (three major peaks). MSCEV sample did not show any distinct peaks. f. RT-PCR analysis of MSC and MSCNV mRNA. MSCs and MSCNVs contain almost the same amount of ACTB, VEGFA and TGFB mRNAs. MSCEV contain too little mRNA for RT-PCR analysis.

In vitro MSCNV treatment promotes the proliferation and migration of skin fibroblasts To quantify the time for which MSCNVs remain in recipient cells after treatment, MSCNVs labeled with lipid (DiI) were applied to skin fibroblasts. Percentages of the fibroblasts that had lipid (DiI) stained MSCNVs changed very little until day 6 of the treatment (Figure S3. FACS analysis of MSCNV treated cells). However, gel electrophoresis of total RNA in MSCNV showed that most of the mRNAs seemed to have degraded after 48h incubation at 37°C (Figure S4. mRNA stability analysis of MSCNVs). Therefore, we conclude that stable components such as lipids and chemical compounds can remain in recipient cells for a long time, whereas unstable and fragile components such as mRNAs quickly lose their activity. To evaluate the ability of MSCNVs to stimulate cell proliferation, MSCNVs or MSCEVs were applied to syngeneic primary skin fibroblasts. The MSCNV-treated cells proliferated more than


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untreated and EV-treated cells (Figure 2 a). The numbers of fibroblasts were highest in MSCNVtreated groups and lowest in untreated groups (Figure 2 b). A scratch assay was performed to confirm in vitro cell migration and proliferation. Scratched cells were washed away with PBS, then the remaining cells were treated with 50 μg/mL of MSCNVs or MSCEVs for 48 h. As the proliferation and migration of the cells become increasingly active, the width of the scratches decreases. When the cells were observed at 24 h and 48 h, the scratches were narrowest in the samples treated with MSCNVs and widest in the untreated samples (Figure 2 c, d). To exclude the proliferation effect in scratch assays, growth of skin cells was arrested by mitomycin-C (MMC) treatment and applied to a scratch assay. Although MMC-treated cells showed lower scratch closure rate than untreated cells, MSCNV treatment still accelerated scratch closure in MMCtreated cells (Figure S5. Scratch closure analysis with Mitomycin-C). Together, these results indicate that MSCNVs can stimulate both the proliferation and migration of recipient skin fibroblasts.


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Figure 2 a. Phase-contrast images of MSCNV and MSCEV treated cells. Bars:100 μm. b. Cell counts of MSCNV and MSCEV treated cells. MSCEVs and MSCNVs showed increased proliferation. Bars = mean + standard deviation. n=3, * p < 0.05. c. Scratch-closure assay images of MSCNV and MSCEV treated cells. White lines: initial scratch boundaries. d. Quantification of scratch-closure assay. Cell free area of MSCEV and MSCNV-treated cells were decreased. Bars: mean + standard deviation. n = 3, * p < 0.05.

MSCNV treatment promotes the expression of proliferation related molecules Proliferation-related protein expressions and signaling pathways of MSCNV-treated cells were examined using western blots. The intensities of the proliferating cell nuclear antigen (PCNA) and


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c-Myc bands were initially almost the same in all groups (MSCNVs, MSC EVs, untreated, but 24 h after treatment, PCNA expression was higher in cells that had been treated with MSCNVs than in cells that had been treated with MSC EVs, and than in untreated cells (Figure 3 a). Phosphorylation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) were also quantified using western blot 2 h after the MSCNV treatments. The MSCNV and EV treated groups showed elevated phosphorylation of STAT3, but only MSCNV-treated groups showed increased phosphorylation of MAPK (Figure 3 b). The expressions of growth factors in MSCNV-treated skin fibroblasts were also examined using real time quantitative PCR (qPCR) analysis. After 24h treatment of MSCNVs, the expressions of VEGF-α and TGF-β in the treated skin fibroblasts were significantly increased compared to those of the untreated cells (Figure 3 c and d). The increase in growth factors in recipient cells might be affected by the growth factor mRNAs that are enclosed in MSCNVs.


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Figure 3 a. Expression of proliferation related proteins. MSCEV treatment increased only PCNA expression but MSCNV treatment increased both c-Myc and PCNA expression after 24 h serum starvation. b. Phosphorylation of proliferation-related signaling molecules. MSCEV treatment increased only the phosphorylation of STAT3 but MSCNV treatment increased the phosphorylation of both MAPK and STAT3. c. TGF-beta expression and d. VEGF-alpha expression of MSCNV-treated skin fibroblasts. Application of MSCNV to skin fibroblasts significantly increased the expression of TGF-beta and VEGF-alpha mRNA. Bars: mean + standard deviation. n = 3, * p

Mesenchymal Stem Cell Engineered Nanovesicles for Accelerated

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