miRNA-BASED PHARMACEUTICAL COMPOSITIONS AND USES THEREOF FOR THE PREVENTION AND THE TREATMENT OF TISSUE DISORDERS
20220409652 · 2022-12-29
Assignee
Inventors
Cpc classification
C12N2320/32
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61P19/04
HUMAN NECESSITIES
International classification
A61K31/7088
HUMAN NECESSITIES
A61P19/04
HUMAN NECESSITIES
Abstract
The present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of (i) at least three miRNAs selected in any one of Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 or Table 12 and (ii) a pharmaceutical acceptable vehicle. The pharmaceutical composition according to the invention may be of therapeutic use for the prevention and/or the treatment of tissue disorders, including, but not limited to skin disorders, bone disorders and/or cartilage disorders.
Claims
1. A pharmaceutical composition comprising (i) a therapeutically effective amount of at least three miRNAs and (ii) a pharmaceutical acceptable vehicle, wherein said at least three miRNAs are selected in a group comprising hsa-miR-210-3p, hsa-miR-409-3p, hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-382-5p, hsa-miR-4485-3p, hsa-miR-4454, hsa-miR-619-5p, hsa-miR-3607-5p, hsa-miR-3613-3p, hsa-miR-664b-5p, hsa-miR-3687, hsa-miR-3653-5p, hsa-miR-664b-3p, and a combination thereof.
2-3. (canceled)
4. The pharmaceutical composition according to claim 1, wherein said at least three miRNAs comprise hsa-miR210-3p and/or hsa-miR-409-3p.
5. The pharmaceutical composition according to claim 1, wherein the composition is desiccated and/or sterilized.
6. A method of treatment or prevention of a tissue disorder, wherein the pharmaceutical composition according to claim 1, is administered to an animal or a human individual.
7. (canceled)
8. The method according to claim 6, wherein said tissue is selected from the group comprising bone tissue, cartilage tissue, skin tissue, muscular tissue, epithelial tissue, endothelial tissue, connective tissue, neural tissue and adipose tissue.
9. The method according to claim 6, wherein the tissue disorder is a bone disorder and/or a cartilage disorder.
10. The method according to claim 6, wherein the tissue disorder is a skin disorder.
11. The method according to claim 6, wherein the tissue disorder is selected in a group comprising aplasia cutis congenita; a burn; a cancer, including a breast cancer, a skin cancer and a bone cancer; a Compartment syndrome (CS); epidermolysis bulbosa; giant congenital nevi; an ischemic muscular injury of lower limbs; a muscle contusion, rupture or strain; a post-radiation lesion; and an ulcer, including a diabetic ulcer, preferably a diabetic foot ulcer; arthritis; bone fracture; bone frailty; Caffey's disease; congenital pseudarthrosis; cranial deformation; cranial malformation; delayed union; infiltrative disorders of bone; hyperostosis; loss of bone mineral density; metabolic bone loss; osteogenesis imperfecta; osteomalacia; osteonecrosis; osteopenia; osteoporosis; Paget's disease; pseudarthrosis; sclerotic lesions; spina bifida; spondylolisthesis; spondylolysis; chondrodysplasia; costochondri tis; enchondroma; hallux rigidus; hip labral tear; osteochondritis dissecans; osteochondrodysplasia; polychondritis; and the likes.
12. The method according to claim 6, wherein the pharmaceutical composition is administered for tissue reconstruction.
13-17. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
[0380] The present invention is further illustrated by the following examples.
Example 1
Production of Exosomal miRNAs (miRNA Cocktails) According to the Invention
[0381] 1. Materials and Methods
[0382] 1.1 Isolation of hASCs
[0383] Human subcutaneous adipose tissues were harvested by lipo-aspiration following Coleman technique in the abdominal region and after informed consent and serologic screening.
[0384] Human adipose tissue-derived stem cells (hASCs) were promptly isolated from the incoming adipose tissue. Lipoaspirate can be stored at +4° C. for 24 to 72 hours or for a longer time at −18° C.
[0385] First, a fraction of the lipoaspirate was isolated for quality control purposes and the remaining volume of the lipoaspirate was measured. Then, the lipoaspirate was digested by a collagenase solution (NB 1, Serva Electrophoresis® GmbH, Heidelberg, Germany) prepared in HBSS (with a final concentration of ˜8 U/mL). The volume of the enzyme solution used for the digestion was the double of the volume of the adipose tissue. The digestion was performed during 50-70 min at 37° C.±1° C. A first intermittent shaking was performed after 15-25 min and a second one after 35-45 min. The digestion was stopped by the addition of MP medium (proliferation medium, or growth medium). The MP medium comprised DMEM medium (4.5 g/L glucose and 4 mM Ala-Gln; Sartorius Stedim Biotech®, Gottingen, Germany) supplemented with 5% human platelet lysate (hPL) (v/v). DMEM is a standard culture medium containing salts, amino acids, vitamins, pyruvate and glucose, buffered with a carbonate buffer and has a physiological pH (7.2-7.4). The DMEM used contained Ala-Gln. Human platelet lysate (hPL) is a rich source of growth factor used to stimulate in vitro growth of mesenchymal stem cells (such as hASCs).
[0386] The digested adipose tissue was centrifuged (500×g, 10 min, 20° C.) and the supernatant was removed. The pelleted Stromal Vascular Fraction (SVF) was re-suspended into MP medium and passed through a 200-500 μm mesh filter. The filtered cell suspension was centrifuged a second time (500×g, 10 min, 20° C.). The pellet containing the hASCs was re-suspended into MP medium. A small fraction of the cell suspension can be kept for cell counting and the entire remaining cell suspension was used to seed one 75 cm.sup.2 T-flask (referred as Passage P0). Cell counting was performed (for information only) in order to estimate the number of seeded cells.
[0387] The day after the isolation step (day 1), the growth medium was removed from the 75 cm.sup.2 T-flask. Cells were rinsed three times with phosphate buffer and freshly prepared MP medium was then added to the flask.
[0388] 1.2 Growth and Expansion of Human Adipose Tissue-Derived Stem Cells
[0389] During the proliferation phase, hASCs were passaged 4 times (P1, P2, P3 and P4) in order to obtain a sufficient amount of cells for the subsequent steps of the process.
[0390] Between P0 and the fourth passage (P4), cells were cultivated on T-flasks and fed with fresh MP medium. Cells were passaged when reaching a confluence ≥70% and ≤100% (target confluence: 80-90%). All the cell culture recipients from 1 batch were passaged at the same time. At each passage, cells were detached from their culture vessel with TrypLE (Select 1×; 9 mL for 75 cm.sup.2 flasks or 12 mL for 150 cm.sup.2 flasks), a recombinant animal-free cell-dissociation enzyme. TrypLe digestion was performed for 5-15 min at 37° C.±2° C. and stopped by the addition of MP medium.
[0391] Cells were then centrifuged (500×g, 5 min, 20° C.), and re-suspended in MP medium. Harvested cells were pooled in order to guaranty a homogenous cell suspension. After resuspension, cells were counted. At passages P1, P2 and P3, the remaining cell suspension was then diluted to the appropriate cell density in MP medium and seeded on larger tissue culture surfaces. At these steps, 75 cm.sup.2 flasks were seeded with a cell suspension volume of 15 mL, while 150 cm.sup.2 flasks were seeded with a cell suspension volume of 30 mL. At each passage, cells were seeded between 0.5×10.sup.4 and 0.8×10.sup.4 cells/cm.sup.2. Between the different passages, culture medium was exchanged every 3-4 days. The cell behavior and growth rate from one donor to another could slightly differ. Hence the duration between two passages and the number of medium exchanges between passages may vary from one donor to another.
[0392] 1.3 Osteogenic Differentiation
[0393] At passage P4 (i.e., the fourth passage), cells were centrifuged a second time, and re-suspended in MD medium (differentiation medium). After resuspension, cells were counted a second time before being diluted to the appropriate cell density in MD medium, and a cell suspension volume of 70 mL was seeded on 150 cm.sup.2 flasks and fed with osteogenic MD medium. According to this method, cells were directly cultured in osteogenic MD medium after the fourth passage. Therefore, osteogenic MD medium was added while cells have not reached confluence.
[0394] The osteogenic MD medium was composed of proliferation medium (DMEM, Ala-Gln, hPL 5%) supplemented with dexamethasone (1 μM), ascorbic acid (0.25 mM) and sodium phosphate (2.93 mM).
[0395] The cell behavior and growth rate from one donor to another could slightly differ. Hence the duration of the osteogenic differentiation step and the number of medium exchanges between passages may vary from one donor to another.
[0396] 1.4 Multi-Dimensional Induction of Cells
[0397] The multi-dimensional induction of ASCs was launched when cells reach a confluence and if a morphologic change appears and if at least one osteoid nodule (i.e., the un-mineralized, organic portion of the bone matrix that forms prior to the maturation of bone tissue) was observed in the flasks.
[0398] a) 3D-Induction in the Presence of Gelatin (NVD002 Biomaterial)
[0399] After being exposed to the osteogenic MD medium, the culture vessels containing the confluent monolayer of adherent osteogenic cells were slowly and homogeneously sprinkled with gelatin particles (Cultispher®-S, Percell Biolytica®, Astorp, Sweden) at a concentration of 1.5 cm.sup.3 for a 150 cm.sup.2 vessel.
[0400] Cells were maintained in MD medium. Regular medium exchanges were performed every 3 to 4 days during the multi-dimensional induction. Those medium exchanges were performed by carefully preventing removal of gelatin particles and developing structure(s). The corresponding biomaterial is referred to as NVD002.
[0401] b) 3D-Induction in the Presence of HA/β-TCP (NVD003 Biomaterial)
[0402] After being exposed to the osteogenic MD medium, the culture vessels containing the confluent monolayer of adherent osteogenic cells were slowly and homogeneously sprinkled with HA/β-TCP particles (in a ratio 60/40): 3 cm.sup.3 for a 150 cm.sup.2 flask (Biomatlante®, France).
[0403] Cells were maintained in MD medium. Regular medium exchanges were performed every 3 to 4 days during the multi-dimensional induction. Those medium exchanges were performed by carefully preventing removal of ceramic material particles and developing structure(s). The corresponding biomaterial is referred to as NVD003. The RNAs' content, in particular the miRNAs' content, was recovered from the obtained biomaterial, which constitutes a miRNAs cocktail.
[0404] 1.5 miRNAs' Content
[0405] a) RNA Extraction
[0406] mRNAs isolation was performed from biopsies. mRNAs were extracted using miRNeasy kit Mastermix (Qiagen®, Hilden, Germany) following the manufacturer's protocol. RNA concentration was determined by Nanodrop (ThermoFisher®, Waltham, Mass., USA). To assess the quality of the samples, 2 μL of RNA was analyzed using RNA pico chip Agilent® Bioanalyzer (Agilent®, Santa Clara, Calif., USA). Three biological replicates were prepared per condition. Long RNA libraries were generated using TruSeq® Stranded Total RNA Library Prep (RS-122-2001, Illumina®, San Diego, Calif., USA) and small RNA libraries using the SMARTer® smRNA-Seq kit (635030, Takara Kusatsu, Shiga, Japan).
[0407] Sequencing was performed using an Illumina NextSeq® 500 (75-pb single-end reads) (Illumina®, San Diego, Calif., USA) generating approximately 26 million reads per long RNA libraries.
[0408] b) Ouantitative RT-PCR (qRT-PCR)
[0409] For quantification of miRNA expression, 50 ng RNA was reverse transcribed into cDNA using qScript miRNA cDNA Synthesis kit (Quanta Biosciences®), and qRT-PCR was conducted in triplicate using Perfecta SYBR Green Super Mix (Quanta Biosciences®). Thermal cycling was performed on an Applied Biosystems 7900 HT detection system (Applied Biosystems®). Data was normalized to miR-16-5p and U6 small nuclear RNA using the Delta-Delta Ct method.
[0410] c) Exosomes Purification
[0411] Exosomes have been isolated by differential centrifugation from culture medium whereby larger “contaminants” are first excluded by pelleting out through increasing speeds of centrifugation before exosomes, small extracellular vesicles and even protein aggregates are pelleted at very high speeds (˜100,000×g).
[0412] Briefly, the culture medium was collected and precleared by centrifugation at 400×g for 5 minutes, then 2,000×g for 20 min at 4° C., followed by centrifugation at 12,000×g for 45 min at 4° C. to eliminate dead cells and cellular debris. Then, the supernatant was passed through a 0.22-μm filter (Millipore®). The supernatant was then ultracentrifuged at 110,000×g for 120 minutes at 4° C., followed by washing of the exosome pellet with phosphate-buffered saline (PBS) at 110,000×g for 120 minutes at 4° C. (Optima XPN-80 Ultracentrifuge, Beckman Coulter®). The supernatant was discarded and the exosome pellet was lysed with Qiazol and stored at −80° C. for further analysis.
[0413] 2. Results
[0414] a) Identification of miRNAs Obtained after 3D-Induction with Gelatin (NVD002)
TABLE-US-00013 TABLE 13 identification of miRNAs from purified exosomes hsa-let-7a-5p hsa-miR-92a-3p hsa-miR-92b-3p hsa-miR-24-2-5p hsa-let-7b-5p hsa-miR-125b-5p hsa-miR-335-5p hsa-miR-26a-2-3p hsa-let-7f-5p hsa-miR-337-3p hsa-let-7f-1-3p hsa-miR-301a-3p hsa-miR-24-3p hsa-miR-93-5p hsa-miR-196b-5p hsa-miR-98-3p hsa-miR-21-5p hsa-miR-409-3p hsa-miR-3613-3p hsa-miR-1273a hsa-miR-23b-3p hsa-miR-199a-3p hsa-miR-23a-5p hsa-miR-28-5p hsa-miR-1273g-3p hsa-miR-145-5p hsa-miR-374b-5p hsa-miR-34a-3p hsa-miR-574-3p hsa-miR-30a-3p hsa-miR-660-5p hsa-miR-425-3p hsa-miR-25-3p hsa-miR-382-5p hsa-miR-186-5p hsa-miR-505-3p hsa-let-7e-5p hsa-miR-19b-3p hsa-miR-454-3p hsa-miR-34b-3p hsa-miR-214-3p hsa-miR-210-3p hsa-miR-10a-5p hsa-miR-361-3p hsa-miR-199a-5p hsa-miR-619-5p hsa-miR-495-3p hsa-miR-10b-5p hsa-miR-196a-5p hsa-miR-17-5p hsa-miR-425-5p hsa-miR-1306-5p hsa-miR-199b-5p hsa-miR-193a-5p hsa-miR-2053 hsa-miR-22-5p hsa-miR-221-3p hsa-miR-320b hsa-miR-5096 hsa-miR-378a-3p hsa-miR-424-5p hsa-miR-193b-5p hsa-miR-494-3p hsa-miR-411-5p hsa-miR-23a-3p hsa-miR-320a hsa-miR-27a-3p hsa-miR-505-5p hsa-let-7c-5p hsa-miR-151a-3p hsa-miR-4449 hsa-miR-664a-3p hsa-miR-199b-3p hsa-let-7a-3p hsa-miR-532-3p hsa-miR-26a-5p hsa-miR-191-5p hsa-miR-30e-3p hsa-miR-532-5p hsa-miR-377-3p hsa-miR-574-5p hsa-miR-22-3p hsa-miR-126-5p hsa-miR-485-3p hsa-miR-424-3p hsa-miR-99b-5p hsa-miR-30c-5p hsa-miR-590-3p hsa-miR-423-5p hsa-miR-625-3p hsa-miR-130b-3p hsa-miR-99a-3p hsa-miR-342-3p hsa-miR-4668-5p hsa-miR-136-3p hsa-miR-143-3p hsa-let-7d-3p hsa-miR-29b-3p hsa-miR-15b-3p hsa-miR-26b-3p hsa-miR-130a-3p hsa-miR-423-3p hsa-miR-29b-1-5p hsa-miR-3607-5p hsa-miR-3184-3p hsa-miR-376c-3p hsa-miR-99b-3p hsa-miR-3651 hsa-miR-222-3p hsa-let-7b-3p hsa-miR-127-3p hsa-miR-374a-3p hsa-let-7g-5p hsa-miR-3074-5p hsa-miR-134-5p hsa-miR-376a-3p hsa-miR-125a-5p hsa-miR-98-5p hsa-miR-324-5p hsa-miR-485-5p hsa-let-7d-5p hsa-miR-185-5p hsa-miR-3605-3p hsa-miR-103b hsa-miR-29a-3p hsa-miR-19a-3p hsa-miR-101-3p hsa-miR-126-3p hsa-let-7i-5p hsa-miR-34a-5p hsa-miR-103a-3p hsa-miR-149-5p hsa-miR-146b-5p hsa-miR-374c-3p hsa-miR-1246 hsa-miR-193b-3p hsa-miR-4454 hsa-miR-181a-5p hsa-miR-138-5p hsa-miR-223-3p hsa-miR-28-3p hsa-miR-328-3p hsa-miR-190a-5p hsa-miR-340-3p hsa-miR-874-3p hsa-miR-7847-3p hsa-miR-6724-5p hsa-miR-369-5p
TABLE-US-00014 TABLE 14 identification of cellular miRNAs hsa-let-7a-5p hsa-miR-210-3p hsa-miR-29b-3p hsa-miR-30e-3p hsa-let-7b-5p hsa-miR-3184-3p hsa-miR-92a-3p hsa-miR-320a hsa-miR-24-3p hsa-let-7d-5p hsa-miR-193b-5p hsa-miR-361-3p hsa-miR-199a-5p hsa-miR-25-3p hsa-miR-181a-5p hsa-miR-151a-3p hsa-miR-214-3p hsa-miR-193a-5p hsa-miR-30c-5p hsa-miR-154-5p hsa-let-7f-5p hsa-miR-199a-3p hsa-miR-664b-3p hsa-miR-664a-5p hsa-miR-3607-5p hsa-miR-29a-3p hsa-miR-27a-3p hsa-miR-92b-3p hsa-miR-199b-3p hsa-miR-342-3p hsa-miR-320b hsa-miR-1291 hsa-let-7e-5p hsa-miR-130a-3p hsa-miR-3651 hsa-miR-103b hsa-miR-1273g-3p hsa-miR-30a-3p hsa-miR-664b-5p hsa-miR-34a-3p hsa-miR-125a-5p hsa-miR-145-5p hsa-miR-664a-3p hsa-miR-140-5p hsa-miR-21-5p hsa-miR-28-3p hsa-miR-98-5p hsa-miR-3609 hsa-let-7i-5p hsa-miR-93-5p hsa-miR-146b-5p hsa-miR-374c-3p hsa-miR-125b-5p hsa-miR-34a-5p hsa-miR-337-3p hsa-miR-10a-5p hsa-let-7g-5p hsa-miR-222-3p hsa-miR-4449 hsa-miR-22-3p hsa-miR-191-5p hsa-miR-3074-5p hsa-miR-6516-3p hsa-miR-4668-5p hsa-miR-574-3p hsa-miR-424-5p hsa-let-7i-3p hsa-miR-24-2-5p hsa-miR-199b-5p hsa-miR-424-3p hsa-miR-103a-3p hsa-miR-29b-1-5p hsa-miR-423-5p hsa-miR-328-3p hsa-miR-324-5p hsa-miR-335-5p hsa-miR-574-5p hsa-miR-17-5p hsa-miR-660-5p hsa-miR-425-5p hsa-miR-23b-3p hsa-miR-23a-3p hsa-miR-185-5p hsa-miR-4461 hsa-miR-196a-5p hsa-let-7d-3p hsa-miR-374b-5p hsa-miR-127-3p hsa-let-7c-5p hsa-miR-423-3p hsa-miR-409-3p hsa-miR-196b-5p hsa-miR-221-3p hsa-miR-382-5p hsa-miR-619-5p hsa-miR-3613-5p hsa-miR-3653-5p hsa-miR-19b-3p hsa-miR-99b-5p hsa-miR-376c-3p hsa-miR-99b-3p hsa-miR-663b hsa-miR-495-3p hsa-miR-454-3p
TABLE-US-00015 TABLE 15 Level of exosomal miRNAs from the NVD002 biomaterial as compared to a 2D culture miRNA logFC FC logCPM PValue FDR hsa-miR-3687 −5.4340 −43.2330 9.986598816 6.14E−08 3.38E−05 hsa-miR-664b-5p −6.7300 −106.1558 9.571690269 1.19E−06 0.00032614 hsa-miR-210-3p 3.3558 10.2381 10.48338867 2.04E−06 0.00035487 hsa-miR-4449 −2.9142 −7.5382 11.07025445 2.58E−06 0.00035487 hsa-miR-3651 −4.4165 −21.3551 11.30176655 1.47E−05 0.00140376 hsa-miR-663a −5.5373 −46.4407 10.73936527 1.53E−05 0.00140376 hsa-miR-664b-3p −7.2564 −152.9025 10.0417161 2.26E−05 0.00177881 hsa-miR-3653-5p −3.7408 −13.3696 9.73359759 5.05E−05 0.00347481 hsa-miR-664a-3p −2.8640 −7.2806 10.34200935 0.000189789 0.01159819 hsa-miR-3648 −3.5109 −11.4000 9.789761883 0.000273373 0.01503553 hsa-miR-619-5p 1.9219 3.7892 10.62269524 0.002222957 0.11114785 hsa-miR-181a-5p 2.5395 5.8140 9.387142262 0.002792503 0.12798972 hsa-miR-409-3p −1.3868 −2.6151 12.81792844 0.00335429 0.14191227 hsa-let-7a-3p 6.5703 95.0297 9.606951776 0.004329701 0.17009541 hsa-miR-4454 2.6272 6.1784 11.53331453 0.004846818 0.17771667 hsa-let-7i-5p 1.4660 2.7626 11.86103511 0.006780515 0.23308021 hsa-miR-335-5p 3.5260 11.5196 9.315665201 0.007991541 0.25854987 hsa-miR-1246 5.0336 32.7554 8.539445396 0.009781646 0.29888364 hsa-miR-6516-5p −4.1176 −17.3593 9.073414111 0.011915311 0.34491691 hsa-miR-3607-5p −2.0844 −4.2410 9.626131484 0.012915156 0.35516678 hsa-let-7e-5p 1.3793 2.6014 13.88843995 0.016482404 0.43168201 hsa-miR-25-3p −1.0699 −2.0993 14.99237678 0.020116155 0.50290388 hsa-miR-374c-3p 2.1608 4.4719 9.494278145 0.0222919 0.53306717 hsa-miR-29b-3p 1.5768 2.9832 9.845249887 0.025174313 0.56328252 hsa-let-7b-3p −1.4233 −2.6820 10.95150956 0.025603751 0.56328252 hsa-miR-23b-3p 1.2910 2.4470 14.73760912 0.028045493 0.5796539 hsa-miR-3613-3p −2.0169 −4.0472 10.92938585 0.028455737 0.5796539 hsa-miR-138-5p −1.5674 −2.9637 9.777277597 0.040019769 0.78610261 hsa-miR-6516-3p −2.0847 −4.2420 8.826423155 0.042870909 0.81306896
TABLE-US-00016 TABLE 16 Level of cellular miRNAs from the NVD002 biomaterial as compared to a 2D culture miRNA logFC FC logCPM PValue FDR hsa-miR-210-3p 6.4208 85.6764 12.48420101 6.80E−18 2.42E−15 hsa-miR-4485-3p −4.0409 −16.4603 11.3547262 8.18E−11 1.46E−08 hsa-miR-24-3p 1.1078 2.1551 16.03988644 2.58E−05 0.0030666 hsa-miR-409-3p −2.2145 −4.6413 11.39958036 0.000257967 0.02295903 hsa-let-7i-5p 1.1353 2.1966 13.67239011 0.000499245 0.03554627 hsa-miR-382-5p −1.2517 −2.3813 11.92251263 0.00225027 0.133516 hsa-miR-214-3p 0.8790 1.8391 15.62352032 0.003282859 0.16695684 hsa-miR-199b-5p 1.2889 2.4435 13.44753159 0.006034794 0.24998011 hsa-miR-199a-5p 0.8790 1.8391 15.68100144 0.006319722 0.24998011 hsa-miR-3074-5p 1.2659 2.4048 11.37814998 0.009651574 0.34359603 hsa-miR-361-3p 2.8526 7.2232 9.80277522 0.013351705 0.43210973 hsa-miR-6723-5p −4.6621 −25.3195 9.311990262 0.015695848 0.46564349 hsa-miR-130a-3p 1.7314 3.3205 12.0290808 0.017837723 0.48847918 hsa-miR-663a −2.7768 −6.8533 9.692314667 0.019868045 0.505216 hsa-miR-3607-5p 0.6038 1.5197 14.71277969 0.021552995 0.5115244 hsa-miR-660-5p 1.5747 2.9787 10.15389819 0.025273272 0.5623303 hsa-miR-196b-5p −1.6406 −3.1181 10.12403868 0.028246436 0.5915136 hsa-miR-4449 −1.1855 −2.2744 11.22700361 0.031038083 0.61386431 hsa-miR-342-3p 0.9434 1.9230 12.22137602 0.038570501 0.70780213 hsa-miR-221-3p −0.7419 −1.6723 13.67334394 0.039764165 0.70780213
[0415] b) Identification of miRNAs Obtained after 3D-Induction with HA/β-TCP (NVD003)
TABLE-US-00017 TABLE 17 identification of miRNAs from purified exosomes hsa-let-7a-5p hsa-let-7i-5p hsa-miR-660-5p hsa-miR-6832-3p hsa-let-7b-5p hsa-miR-409-3p hsa-miR-664a-3p hsa-miR-146a-5p hsa-miR-24-3p hsa-miR-210-3p hsa-miR-185-5p hsa-miR-16-2-3p hsa-miR-21-5p hsa-miR-199a-3p hsa-miR-3651 hsa-miR-181b-5p hsa-let-7f-5p hsa-miR-30a-3p hsa-miR-495-3p hsa-miR-26a-2-3p hsa-miR-574-3p hsa-miR-320b hsa-let-7a-3p hsa-miR-376a-3p hsa-miR-23b-3p hsa-miR-193a-5p hsa-miR-28-5p hsa-miR-539-5p hsa-miR-1273g-3p hsa-miR-382-5p hsa-miR-99b-3p hsa-miR-708-5p hsa-miR-25-3p hsa-miR-423-3p hsa-miR-103a-3p hsa-miR-98-3p hsa-miR-199a-5p hsa-miR-17-5p hsa-miR-19a-3p hsa-miR-1237-5p hsa-miR-196a-5p hsa-miR-19b-3p hsa-miR-126-5p hsa-miR-223-3p hsa-miR-214-3p hsa-miR-92b-3p hsa-miR-2053 hsa-miR-532-5p hsa-miR-125a-5p hsa-miR-320a hsa-miR-29b-1-5p hsa-miR-542-3p hsa-miR-221-3p hsa-miR-3074-5p hsa-miR-3648 hsa-miR-663a hsa-miR-222-3p hsa-miR-376c-3p hsa-miR-374a-3p hsa-miR-101-3p hsa-let-7e-5p hsa-let-7b-3p hsa-miR-454-3p hsa-miR-143-3p hsa-miR-191-5p hsa-miR-625-3p hsa-miR-532-3p hsa-miR-21-3p hsa-miR-199b-3p hsa-miR-99b-5p hsa-miR-136-3p hsa-miR-224-5p hsa-miR-342-3p hsa-miR-34a-5p hsa-miR-361-3p hsa-miR-26a-5p hsa-miR-23a-3p hsa-miR-5096 hsa-miR-1246 hsa-miR-27a-5p hsa-miR-424-3p hsa-miR-30e-3p hsa-miR-130b-3p hsa-miR-324-5p hsa-miR-28-3p hsa-miR-22-3p hsa-miR-134-5p hsa-miR-340-3p hsa-let-7g-5p hsa-miR-151a-3p hsa-miR-154-5p hsa-miR-379-5p hsa-miR-92a-3p hsa-miR-186-5p hsa-miR-34a-3p hsa-miR-409-5p hsa-miR-424-5p hsa-miR-193b-5p hsa-miR-576-5p hsa-miR-543 hsa-let-7d-3p hsa-miR-328-3p hsa-miR-874-3p hsa-miR-5787 hsa-miR-4454 hsa-miR-4449 hsa-miR-100-5p hsa-miR-6089 hsa-miR-146b-5p hsa-miR-27a-3p hsa-miR-103b hsa-miR-127-3p hsa-miR-423-5p hsa-miR-30c-5p hsa-miR-1273a hsa-miR-149-5p hsa-miR-29a-3p hsa-miR-494-3p hsa-miR-1306-5p hsa-miR-181c-5p hsa-miR-574-5p hsa-miR-98-5p hsa-miR-138-5p hsa-miR-193b-3p hsa-miR-199b-5p hsa-miR-10a-5p hsa-miR-15b-3p hsa-miR-222-5p hsa-miR-125b-5p hsa-miR-29b-3p hsa-miR-26b-3p hsa-miR-3613-5p hsa-miR-3184-3p hsa-miR-374b-5p hsa-miR-10b-5p hsa-miR-365b-3p hsa-let-7c-5p hsa-miR-335-5p hsa-miR-22-5p hsa-miR-3960 hsa-miR-33 7-3p hsa-miR-374c-3p hsa-miR-3613-3p hsa-miR-485-3p hsa-let-7d-5p hsa-miR-425-5p hsa-miR-655-3p hsa-miR-6087 hsa-miR-145-5p hsa-miR-181a-5p hsa-miR-7-1-3p hsa-miR-92a-1-5p hsa-miR-93-5p hsa-miR-196b-5p hsa-miR-23a-5p hsa-miR-4668-5p hsa-miR-619-5p hsa-let-7f-1-3p hsa-miR-24-2-5p hsa-miR-3605-3p hsa-miR-130a-3p
TABLE-US-00018 TABLE 18 identification of cellular miRNAs hsa-let-7a-5p hsa-miR-3653-5p hsa-miR-98-5p hsa-miR-28-5p hsa-let-7b-5p hsa-miR-342-3p hsa-miR-664a-3p hsa-miR-10a-5p hsa-miR-24-3p hsa-miR-28-3p hsa-miR-92b-3p hsa-miR-151a-3p hsa-let-7f-5p hsa-miR-23b-3p hsa-miR-4449 hsa-miR-30e-3p hsa-miR-199a-5p hsa-let-7c-5p hsa-miR-320a hsa-miR-324-5p hsa-miR-214-3p hsa-miR-222-3p hsa-miR-181a-5p hsa-miR-495-3p hsa-miR-3607-5p hsa-miR-29a-3p hsa-miR-3651 hsa-miR-576-5p hsa-miR-125a-5p hsa-miR-92a-3p hsa-miR-185-5p hsa-miR-625-3p hsa-miR-199b-3p hsa-miR-30a-3p hsa-miR-664b-5p hsa-miR-671-5p hsa-miR-125b-5p hsa-miR-424-3p hsa-miR-196b-5p hsa-miR-1271-5p hsa-miR-21-5p hsa-miR-423-3p hsa-miR-27a-3p hsa-miR-186-5p hsa-let-7e-5p hsa-miR-34a-5p hsa-miR-29b-3p hsa-miR-23a-5p hsa-let-7i-5p hsa-miR-424-5p hsa-miR-664b-3p hsa-miR-3613-5p hsa-let-7g-5p hsa-miR-145-5p hsa-miR-99b-5p hsa-miR-376c-3p hsa-miR-574-3p hsa-miR-328-3p hsa-miR-103a-3p hsa-miR-409-3p hsa-miR-574-5p hsa-miR-3074-5p hsa-miR-6516-3p hsa-miR-4461 hsa-miR-191-5p hsa-let-7d-3p hsa-miR-22-3p hsa-miR-454-3p hsa-miR-196a-5p hsa-miR-93-5p hsa-miR-26a-5p hsa-miR-6724-5p hsa-miR-221-3p hsa-miR-23a-3p hsa-miR-103b hsa-let-7b-3p hsa-miR-25-3p hsa-miR-19b-3p hsa-miR-1291 hsa-miR-190a-5p hsa-miR-423-5p hsa-miR-146b-5p hsa-miR-425-5p hsa-miR-26b-3p hsa-miR-210-3p hsa-miR-320b hsa-miR-22-5p hsa-miR-3609 hsa-miR-1273g-3p hsa-miR-337-3p hsa-miR-374c-3p hsa-miR-411-5p hsa-let-7d-5p hsa-miR-17-5p hsa-let-7i-3p hsa-miR-425-3p hsa-miR-199b-5p hsa-miR-130a-3p hsa-miR-374b-5p hsa-miR-4485-3p hsa-miR-199a-3p hsa-miR-193b-5p hsa-miR-455-3p hsa-miR-30c-5p hsa-miR-193a-5p hsa-miR-382-5p hsa-miR-532-3p hsa-miR-619-5p hsa-miR-3184-3p
TABLE-US-00019 TABLE 19 Level of exosomal miRNAs from the NVD003 biomaterial as compared to a 2D culture miRNA logFC FC logCPM PValue FDR hsa-miR-210-3p 3.8329 14.2502 11.0073 5.86E−07 0.00016341 hsa-miR-3687 −4.2961 −19.6462 10.1302 4.12E−07 0.00016341 hsa-miR-4454 3.3561 10.2401 12.1672 1.14E−06 0.00021296 hsa-miR-3653-5p −4.4413 −21.7264 9.7928 3.76E−06 0.00052506 hsa-miR-664b-5p −4.2939 −19.6152 9.7186 9.08E−06 0.00101337 hsa-miR-619-5p 2.7545 6.7485 11.3607 0.00016339 0.0130247 hsa-miR-664b-3p −4.9289 −30.4622 10.1914 0.00014999 0.0130247 hsa-miR-3613-3p −3.0938 −8.5377 10.8210 0.00105987 0.07392561 hsa-miR-409-3p −1.5541 −2.9365 12.8917 0.00139377 0.08641401 hsa-miR-3607-5p −2.9445 −7.6981 9.6272 0.00178514 0.0996108 hsa-miR-1246 5.1208 34.7953 8.6663 0.00292854 0.14855699 hsa-miR-3609 −4.9838 −31.6427 8.4767 0.00415237 0.19308534 hsa-miR-222-3p 1.4242 2.6837 13.2032 0.0053805 0.23094758 hsa-miR-181a-5p 2.3137 4.9717 9.3588 0.00612351 0.24406574 hsa-miR-6832-3p 4.7957 27.7766 8.5024 0.01014361 0.37734231 hsa-miR-335-5p 3.3247 10.0198 9.3569 0.01119602 0.39046125 hsa-let-7a-3p 5.8907 59.3317 9.0729 0.01316075 0.40798328 hsa-miR-28-3p 1.2784 2.4258 12.7300 0.01298217 0.40798328 hsa-miR-663a −3.4819 −11.1732 10.9716 0.01441164 0.42324701 hsa-miR-19a-3p −1.6552 −3.1497 10.3578 0.01740016 0.46234701 hsa-miR-3651 −2.7857 −6.8957 11.5575 0.01725774 0.46234701 hsa-miR-125a-5p 1.2160 2.3231 13.5469 0.0201696 0.51157428 hsa-miR-374c-3p 2.1030 4.2963 9.5512 0.02120255 0.51439235 hsa-miR-4668-5p −1.6714 −3.1853 10.65127 0.02283107 0.53082232 hsa-miR-301a-3p −2.8147 −7.0361 8.9635 0.02430933 0.54258424 hsa-miR-664a-3p −1.7910 −3.4606 10.6389 0.02619156 0.56211116 hsa-miR-485-5p −2.0114 −4.0317 9.0528 0.03141568 0.64925748 hsa-miR-382-5p −1.0946 −2.1355 12.0209 0.03304208 0.65848152 hsa-miR-4449 −1.5489 −2.9260 11.4255 0.03601783 0.68391665 hsa-miR-138-5p −1.5886 −3.0076 9.8986 0.03676971 0.68391665 hsa-miR-181c-5p 4.1112 17.2823 8.2063 0.04528724 0.81517026 hsa-miR-374b-5p 1.7817 3.4383 9.6939 0.04793018 0.83578254
TABLE-US-00020 TABLE 20 Level of cellular miRNAs from the NVD003 biomaterial as compared to a 2D culture miRNA logFC FC logCPM PValue FDR hsa-miR-210-3p 6.4907 89.9288 12.5959 3.65E−17 1.29E−14 hsa-miR-4485-3p −3.7281 −13.2516 11.4563 3.29E−09 5.82E−07 hsa-miR-409-3p −3.3163 −9.9616 11.31065 3.00E−06 0.00035375 hsa-miR-382-5p −1.6105 −3.05363 11.8932 0.00020693 0.01831299 hsa-miR-24-3p 0.9484 1.9298 16.0201 0.00036741 0.0260129 hsa-miR-93-5p −1.5223 −2.8725 12.3200 0.00054198 0.03197659 hsa-let-7i-5p 1.1389 2.2021 13.7506 0.00119986 0.06067875 hsa-miR-3074-5p 1.2618 2.3980 11.4482 0.00741164 0.32796499 hsa-let-7c-5p −0.9293 −1.9044 13.0382 0.01316331 0.47049582 hsa-miR-154-5p −2.0724 −4.2061 10.1415 0.01329084 0.47049582 hsa-miR-6723-5p −4.7630 −27.1540 9.3165 0.01570223 0.50532623 hsa-miR-671-5p 4.2840 19.4815 9.3994 0.03498742 1
Example 2
Skin Wound Reconstruction Properties of NVD002-Derived Exosomes
[0416] The potential functional impact of NVD002-derived exosomes was studied in vitro on one model of cell line: HDFa (human dermal fibroblast).
[0417] NVD002-derived exosomes (ASCs osteo-differentiated and 3D-induced in the presence of gelatin, as in example 1) from 3 donors were co-incubated in 96-wells plates with HDFa cell line at 2.5 and 25 μg/ml for up to 72 h at 37° C., 5% CO.sub.2 under normoxia (21% O.sub.2) or hypoxia (1% O.sub.2). A cell viability test (CellTiter-Glo Cell viability Assay) was performed after 30 minutes to 48 h of co-incubation, at minimum 5 different time points, to evaluate the proliferation of HDFa. The CellTiter-Gloe Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. Experiments were performed in triplicate.
[0418] Statistically significant differences between groups (with normal distribution) were tested by paired t-test and two-way analysis of variance with the Bonferroni post hoc test. Non-normal distributions of data were analyzed using the Kruskal-Wallis test. Statistical tests were performed with Prism GraphPad 2 (NIH). A P value<0.05 was considered significant.
[0419] Proliferation curves of HDFa cells cultured with NVD002-derived exosomes under normoxia (curves 2 and 3 of
[0420] Linear regression of the progression speed curves was calculated. Higher progression speed rates were found for HDFa cells cultured with exosomes, at both 2.5 and 25 μg/ml. A significant higher viability was observed for HDFa cells co-cultured with NVD002-derived exosomes at 2.5 and 2.5 μg/ml at 24 h and 32 h/48 h of incubation of incubation (p<0.01). (
[0421] Proliferation curves of HDFa cells cultured with NVD002-derived exosomes under hypoxia (curves 2 and 3 of
[0422] Linear regression of the progression speed curves was calculated. higher proliferation rates were found for HDFa cells cultured with exosomes, at both 2.5 and 25 μg/ml. A significant higher viability was observed for HDFa cells co-cultured with NVD002-derived exosomes at 2.5 and 2.5 μg/ml at 24 h and 32 h/48 h of incubation of incubation (p<0.01). (
[0423] In conclusion, NVD002-derived exosomes can speed the proliferation of human dermal fibroblast cell lines in vitro. These results suggest that skin repair, including, e.g., the diabetic wound healing, may be achieved with NVD002-derived exosomes.
Example 3
In Vitro Evaluation of the Inhibition of Osteoclastogenesis
[0424] 1. Impact of NVD003-Derived Exosomes (miRNAs Cocktail) on the Inhibition of Osteoclasts Maturation and Activity
[0425] 1.1 Impact of the NVD003-Derived Exosomes on the Differentiation of Osteoclasts Precursors
[0426] a) Osteoclastogenesis in Presence of RANKL
[0427] An in vitro osteoclast differentiation protocol from human monocytes was used. Human CD14+ monocytes were isolated from peripheral blood of healthy volunteers, obtained in agreement with the “Etablissement Francais du Sang”.
[0428] Following isolation of peripheral blood mononuclear cells by Ficoll-Hypaque centrifugation, monocytes (CD14+ cells) were sorted (MACS®, MiltenyiBiotec). Freshly isolated precursors were differentiated into osteoclasts in the presence of M-CSF and RANKL (RANKL medium, “plus RANKL” control). Cells in medium without RANKL serve as a negative control (M-CSF medium, “no RANKL” control). The differentiation time was 8 days. The osteoclatogenesis was carried out in 96-well culture plates.
[0429] At D0, precursors (CD14+) were seeded and incubated for 2 hours (minimum time for cell attachment) in medium supplemented with 1% FBS, 25 ng/mL human MCSF+/−100 ng/mL human RANKL and exosomes were added at 50 and 100 μg/ml in 24-well culture plates. Medium was changed at day 4 and day 7. All treatments and controls were carried out in triplicate.
[0430] A TRAP staining was performed at day 8. The number of TRAP-positive cells containing more than three nuclei was determined in each well.
[0431] As shown in
[0432] b) Osteoclastogenesis in Absence of RANKL+/−Sclerostin
[0433] Human CD14+ monocytes were isolated from peripheral blood of healthy volunteers, obtained in agreement with the “Etablissement Français du Sang”. Following isolation of peripheral blood mononuclear cells by Ficoll-Hypaque centrifugation, monocytes (CD14+ cells) were sorted (MACS®, Miltenyi Biotec). Freshly isolated precursors (CD14+) were cultivated in M-CSF medium (medium supplemented with 1% FBS and 25 ng/mL M-CSF) with or without exosomes and/or sclerostin. Cells in RANKL medium (medium supplemented with 1% FBS, 25 ng/mL M-CSF and 100 ng/mL RANKL) served as positive control. The osteoclatogenesis was carried out in 96-well cell culture plates. At D0, precursors (CD14+) were seeded and incubated for 2 hours (minimum time for cell attachment) in 50 μL of medium supplemented with 1 FBS, 25 ng/mL human M-CSF+/−100 ng/mL human RANKL. Then, exosomes and/or sclerostin were added.
[0434] All treatments and controls were carried out in duplicate. Media (and treatments) were changed at day 4 and day 7. A TRAP staining was performed at day 8. The number of TRAP-positive cells containing more than three nuclei was determined in each well.
[0435] In the absence of RANKL, no osteoclast was formed. No osteoclast was formed in the presence of sclerostin at 10 ng/mL or 100 ng/mL. The combination of exosomes and sclerostin was not more able to induce osteoclast formation in the absence of RANKL.
[0436] 1.2 Impact of the NVD003-Derived Exosomes on the Mature Osteoclasts (Cytotoxicity)
[0437] Human CD14+ monocytes were isolated from peripheral blood of healthy volunteers, obtained in agreement with the “Etablissement Français du Sang”. Osteoclast precursor cells were isolated from the peripheral blood. Following isolation of peripheral blood mononuclear cells by Ficoll-Hypaque centrifugation, monocytes (CD14+ cells) were sorted (MACS®, Miltenyi Biotec). Freshly isolated precursors were differentiated into osteoclasts in the presence of M-CSF and RANKL (“plus RANKL” control) for 5 to 6 days (depending on the donor of CD14+ cells). Cells in medium without RANKL served as a negative control (“no RANKL” control). The differentiation time was 8 days. The osteoclatogenesis was carried out in 96-well culture plates.
[0438] At D0, precursors (CD14+) were seeded and incubated for 2 hours (minimum time for cell attachment) in 50 μL of medium supplemented with 1 FBS, 25 ng/mL human M-CSF+/−100 ng/mL human RANKL.
[0439] When the multinucleated cells were observed in the positive control, medium was renewed. All treatments and controls were carried out in triplicate.
[0440] 48 hours after the addition of the NVD003-derived exosomes, a TRAP staining was performed. The number of TRAP-positive cells containing more than three nuclei was determined in each well.
[0441] As shown in
[0442] 2. Impact of NVD003-Derived Exosomes (miRNA Cocktail) on the Promotion of Osteogenesis
[0443] 2.1 Impact of NVD003-Derived Exosomes on ASCs Osteodifferentiation
[0444] Adipose tissue-derived stem cells (ASCs) at passage 5 (P5) were placed in 96 wells plates in 0.1 mL of proliferation medium (MP) (in the presence of 5% hPL) for about 2 days. Then, the MP was removed and the cells were rinsed 2-fold with PBS. Cells were placed in proliferation medium (MP) or in osteo-differentiation medium (MD), MD+sclerostin (SCL) 100 ng/ml or MD+sclerostin (SCL) 100 ng/ml+NVD003-derived exosomes 100 μg/ml, for 10 days with medium change after 5 days. In addition, cells were placed in proliferation medium (MP) as negative control.
[0445] After 10 days of culture, cells were placed in Qiazol lysis reagent (Qiagen®, Hilden, Germany) for RNA isolation for qRT-PCR for osteogenic genes. Total RNA was extracted from cell lysates. RNAs were purified using Rneasy mini kit (Qiagen®, Hilden, Germany) with an additional on column DNase digestion according to the manufacturer's instruction. Quality and quantity of RNA were determined using a spectrophotometer (Spectramax 190, Molecular Devices®, California, USA). cDNA was synthesized from 0.5 μg of total RNA using RT.sup.2 RNA first strand kit (Qiagene, Hilden, Germany) for osteogenic and angiogenic genes expression profiles though customized PCR arrays (Customized Human Osteogenic and angiogenic RT.sup.2 Profiler Assay—Qiagen®, Hilden, Germany). The ABI Quantstudio 5 system (Applied Biosystemse) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany) were used for detection of the amplification product. Quantification was obtained according to the ΔΔCT method. The final result of each sample was normalized to the means of expression level of three housekeeping genes (ACTB, B2M and GAPDH). Experiments were performed in triplicate.
[0446]
[0447] Surprisingly, culture of ASCs in MD did not induce the expression of all tested osteogenic genes. An overexpression of osteogenic and angiogenic genes MMP, ITGA1, HIF1a, FGF1, ANG, EDN1, TWIST1 and ICAM1 was found in comparison with ASCs in proliferation medium. No impact of SCL was noted on osteogenic genes expression. In contrast, the coculture of exosomes showed the overexpression of skeletal development factors, such as RUNX2 (
[0448] In conclusion, NVD003-derived exosomes at a concentration of 100 μg/ml can potentiate the expression of osteogenic and angiogenic genes by ASCs in osteo-differentiation medium and in presence of sclerostin (100 ng/ml) in comparison to osteo-differentiation medium alone or osteo-differentiation medium+sclerostin.
[0449] 2.2 Impact of NVD003-Derived Exosomes on Osteoblasts Precursors (BM-MSCs)
[0450] A model of osteoblastogenesis from human mesenchymal stem cells was used. Mesenchymal stem cells were thawed according to the recommendations of the supplier. Cells were seeded and cultured in flask in the medium recommended by the supplier for cell proliferation (RoosterBio®, KT-001).
[0451] Four days after thawing, human MSCs were detached with trypsin-EDTA and counted. The cells were seeded at 3.5.Math.10.sup.4 cells per well and cultured in monolayer in 96-well plates in DMEM medium supplemented with 1% FBS for 4 days (the day of seeding is designated day-4). After 4 days of culture in DMEM medium, cells were placed in basal medium (DMEM 1% FBS+ascorbic acid (50 μg/mL) and b-glycerophosphate (10 mM), differentiation medium (positive control) (DMEM 1% FBS+ascorbic acid (50 μg/mL) and b-glycerophosphate (10 mM), dexamethasone (10-8 M) and a vitamin D3 (10-8 M)) or basal medium and NVD003-derived exosomes (the first day of treatment is “Day 0”). Medium and treatments were changed at day 4, day 7 and day 11.
[0452] As the treatment with exosomes at 75 μg/mL required an approximately 1:3 dilution for the stock solution (of a concentration of approximately 200 μg/mL), a control with PBS diluted at 1:3 in differentiation medium and a control with PBS diluted at 1:3 in basal medium were performed. All treatments and controls were carried out in duplicate.
[0453] Cells were lysed at day 7 and day 14 using Qiazol lysis reagent (Qiagen®, Hilden, Germany). For each condition, the triplicates were pooled to provide 1 cell lysate (in total, 13 cell lysates were collected at day 7 and day 14). The volume of each cell lysate was 250 μL. The cell lysates were analyzed by qRT-PCR. Total RNA was extracted from cell lysates. RNAs were purified using Rneasy mini kit (Qiagen®, Hilden, Germany) with an additional on column DNase digestion according to the manufacturer's instruction. Quality and quantity of RNA were determined using a spectrophotometer (Spectramax 190, Molecular devices®, California, USA). mRNA was concentrated using RNA Clean & Concentrator™-5 kit (ZYMO RESEARCH®, Irvine, USA). cDNA was synthesized from 0.5 μg of total RNA using RT.sup.2 RNA first strand kit (Qiagen®, Hilden, Germany) for osteogenic and angiogenic genes expression profiles though a customized osteogenic and angiogenic RT.sup.2 array (Qiagen®, Hilden, Germany). The ABI Quantstudio 5 system (Applied Biosystems®) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany) were used for detection of the amplification product. Quantification was obtained according to the ΔΔCT method. The final result of each sample was normalized to the means of expression level of three housekeeping genes (ACTB, B2M and GAPDH).
[0454]
[0455] Some genes are clearly induced over the basal condition (proliferation medium) as observed in the positive control (osteo-differentiation medium), such as the skeletal development factors RUNX2 (
[0456] Finally, Leptine expression was not induced after exosomes treatment while it was overexpressed after osteo-differentiation (see
[0457] In conclusion, NVD003-derived exosomes treatment of BM-MSCs, at a concentration ranging from 10 μg/ml to 75 μg/ml for 7 days, shows a similar osteogenic and angiogenic expression as found in BM-MSCs in osteo-differentiation medium, except for HIF1a.
Example 4
In Vitro Effect of NVD002-Derived and NVD003-Derived Exosomes on Cancer Cells
[0458] 1. Material and Methods
[0459] a) Cells and Exosomes
[0460] H143B human osteosarcoma cells were obtained from ATCC® (CRL-8303™), A375 human melanoma cells were obtained from ATCC® (CRL-1619™) and U87 human glioblastoma cells were obtained from ATCC® (HTB-14™). NVD002-derived and NVD003-derived exosomes are obtained as disclosed in Example 1.
[0461] b) Proliferation Assay
[0462] NVD003-derived and NVD002-derived exosomes from 3 donors were co-incubated in 96-wells plates with those three cell lines at 2.5 and 25 μg/ml for up to 72 h at 37° C., 5% CO.sub.2. A cell viability test (using the CellTiter-Glo® Cell viability Assay from PROMEGA®) was performed after 30 minutes to 48 h of co-incubation, at minimum 5 different time points, to evaluate the proliferation of targeted cells. The CellTiter-Glo® Luminescent Cell Viability Assay from PROMEGA® is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells). Experiments were performed in triplicate.
[0463] c) Statistical Analysis
[0464] Statistically significant differences between groups (with normal distribution) were tested by paired t-test and one-way analysis of variance with the Bonferroni post hoc test. Non-normal distributions of data were analyzed using the Kruskal-Wallis test. Statistical tests were performed with Prism GraphPad 2 (NIH). Statistical significance are as follows: *:p<0.05; **: p<0.01; ***: p<0.005; ****: p<0.0001.
[0465] 2. Results
[0466] 2.1 In Vitro Effect of Exosomes on Human Osteosarcoma Cells (H143B)
[0467] a) Effect of NVD002-Derived Exosomes
[0468] Proliferation curves of H143B cells cultured with NVD002-derived exosomes showed a slightly lower level of viability than the control cells cultured without NVD002-derived exosomes. In addition, a more marked effect was noted with the highest dose of NVD002-Exosomes exosomes (25 μg/ml vs 2.5 μg/ml) (
[0469] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with NVD002-derived exosomes, at both 2.5 and 25 μg/ml. A significant lower viability signal was found in cells co-cultured with exosomes at 2.5 and 25 μg/ml at 1, 24 and 32 h of incubation and 2.5 μg/ml at 1, 6, 24 and 32 h of incubation (p<0.01). (
[0470] Although a higher slope was found for cells cultured without NVD002-derived exosomes, it was associated with a higher viability level.
[0471] b) Effect of NVD003-Derived Exosomes
[0472] Proliferation curves of H143B cells cultured with NVD003-derived exosomes showed a slightly lower level of viability than the control cells cultured without exosomes (
[0473] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with NVD003-derived exosomes, at both 2.5 and 25 μg/ml. A significant lower viability signal was found in cells co-cultured with NVD003-derived exosomes at 2.5 at 6, 24, 32 and 48 h of incubation and 25 μg/ml only at 24 h and 48 h of incubation p<0.01). (
[0474] Although a higher slope was found for cells cultured without exosomes, it was associated with a higher viability level.
[0475] c) Conclusion
[0476] In conclusion, NVD002-derived and NVD003-derived exosomes can reduce the proliferation of human osteosarcoma cell lines in vitro. A dose-response effect was observed.
[0477] 2.2 In Vitro Effect of Exosomes on Human Melanoma Cells (A375)
[0478] a) Effect of NVD002-Derived Exosomes
[0479] Although similar profile was found between cells cultured without exosomes and 2.5 μg/ml NVD002-derived exosomes, proliferation curves of A375 cells cultured with 25 μg/ml NVD002-derived exosomes showed a lower level of viability than the control cells cultured without exosomes (
[0480] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with NVD002-derived exosomes, at both 2.5 and 25 μg/ml.
[0481] A significant lower viability signal was found in cells co-cultured with NVD002-derived exosomes at 25 μg/ml at 1, 24, 32 and 48 h of incubation (p<0.01). In addition, a significant lower viability signal was found at 24, 32 and 48 h in cells treated with 25 μg/ml NVD002-derived exosomes vs 2.5 μg/ml (p<0.01) (
[0482] Although a higher slope was found for cells cultured without exosomes, it was associated with a higher viability level.
[0483] b) Effect of NVD003-Derived Exosomes
[0484] Proliferation curves of A375 cells cultured with 2.5 and 25 μg/ml NVD003-derived exosomes showed a lower level of viability than the control cells cultured without exosomes. This effect was more marked at 25 μg/ml NVD003-derived exosomes than 2.5 μg/ml (
[0485] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with NVD003-derived exosomes, at both 2.5 and 25 μg/ml. A significant lower viability signal was found in cells co-cultured with NVD003-derived exosomes at 25 μg/ml at each time point of incubation (p<0.01). A significant lower viability signal was found in cells co-cultured with NVD003-derived exosomes at 2.5 μg/ml at 6, 24, 32 and 48 h (p<0.05). A significant lower viability signal was found in cells co-cultured with NVD003-derived exosomes at 25 μg/ml vs 2.5 μg/ml NVD003-derived exosomes at 1, 24, 32, 48 h (p<0.05) (
[0486] Although a higher slope was found for cells cultured without exosomes, it was associated with a higher viability level.
[0487] c) Conclusion
[0488] In conclusion, NVD002-derived and NVD003-derived exosomes can reduce the proliferation of human melanoma cell lines in vitro. A dose-response effect was observed.
[0489] 2.3 In Vitro Effect of Exosomes on Human Glioblastoma Cells (U87)
[0490] a) Effect of NVD002-Derived Exosomes
[0491] Although similar profile was found between cells cultured without exosomes and 2.5 μg/ml exosomes, proliferation curves of U87 cells cultured with 25 μg/ml exosomes showed a lower level of viability than the control cells cultured without exosomes (
[0492] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with exosomes, at both 2.5 and 25 μg/ml, with a more marked effect at 25 than 2.5 μg/ml. A significant lower viability signal was found in cells co-cultured with exosomes at 2.5 only at 6 and 32 h (p<0.05) and 25 μg/ml at each time of incubation (p<0.0001). In addition, a significant lower viability signal was found for U87 cultured with 25 μg/ml NVD002-Exo vs 2.5 μg/ml at each tested timepoint (p<0.0001) (
[0493] Although a higher slope was found for cells cultured without exosomes, it was associated with a higher viability level.
[0494] b) Effect of NVD003-Exosomes
[0495] Proliferation curves of U87 cells cultured with 2.5 and 25 μg/ml exosomes showed a lower level of viability than the control cells cultured without exosomes. This effect was more marked at 25 μg/ml exosomes than 2.5 μg/ml (
[0496] Linear regression of the proliferation curves was calculated. Lower proliferation rates were found for cells cultured with exosomes, at both 2.5 and 25 μg/ml. A significant lower viability signal was found in cells co-cultured with exosomes at 2.5 μg/ml at 6, 24, 32 and 48 h (p<0.0001). In addition, a significant lower viability signal was found for U87 cultured with 25 μg/ml NVD003-Exo vs 2.5 μg/ml at each tested timepoint (p<0.0001). (
[0497] Although a higher slope was found for cells cultured without exosomes, it was associated with a higher viability level.
[0498] c) Conclusion
[0499] In conclusion, NVD002-derived and NVD003-derived exosomes can reduce the proliferation of human glioblastoma cell lines in vitro.