Method for obtaining extracellular vesicles (EVS) derived from induced pluripotent stem cells (IPSCS) and their use

Abstract

A drug for the treatment of organ fibrosis is disclosed, which was developed on the basis of extracellular vesicles derived from induced pluripotent stem cells cultured in conditions of physiological hypoxia.

Description

[0001] Method for obtaining extracellular vesicles (EVs) derived from induced pluripotent stem cells (iPSCs) and their use

[0002] The present invention relates to a drug for the treatment of organ fibrosis, which is developed based on extracellular vesicles (EVs) derived from induced pluripotent stem cells (iPSCs) cultured under physiological hypoxia (5% O2).

[0003] Fibrosis, otherwise known as fibrotic scarring, develops in response to existing chronic inflammation in the body. As a consequence, there is then an increased secretion of cytokines and growth factors, including transforming growth factor beta (TGF- ). This factor is the main activator of stromal cells, such as fibroblasts or stellate cells. After binding to a receptor (TGFBR1 / 2), the TGF-P factor activates an intracellular signalling cascade that initiates the process of fibrosis (Leask A, et al. FASEB J. 2004, 18:816-827). A phenotypic transition of fibroblasts into myofibroblasts (fibroblast-to-myofibroblast transition; FMT) then occurs. Activated myofibroblasts are characterised by increased proliferation and production of extracellular matrix (ECM) proteins, including collagen and fibronectin (Weiskirchen R, et al. Mol Aspects Med. 2019, 65:2-15). These cells also show increased expression of the alphasmooth muscle actin (a-SMA) protein, which is a marker of myofibroblasts (Li B, J Tissue Viability. 2011, 20:108-120). The deposited proteins form a rigid scaffold around the cells that produce them, leading to a stiffening of the microenvironment, which prevents the cells from functioning properly, resulting in cell death. In effect, only the rigid protein matrix remains, forming scar tissue. Organ failure then occurs. Fibrotic diseases are estimated to cause as many as 45% of all deaths worldwide. Thus, they represent a serious health and socio-economic problem (Henderson NC et al. Nature. 2020; 587:555-566).

[0004] To date, no effective cure for fibrotic disease has been developed. Current treatments are based on the elimination of the cause inducing the chronic inflammation (e.g. abstinence in pancreatitis or treatment of hypertriglyceridaemia; treatment of chronic hepatitis B or C), the use of, for example, corticosteroids and drugs that suppress the immune system, or even the removal of part or all of the organ affected by the fibrosis.

[0005] Among the available documents related to the treatment of organ fibrosis, there are entries mentioning the use of N-cadherin protein neutralising antibodies (EP2830659A1), inhibitors targeting proteins: P- catenin (WO2014061827A1); STAT3 (W02009059425A1), aldehyde dehydrogenase (US20170042838A1); CSF-1R (WO2015028454A2) and other compounds, including polysaccharides, gluco / galactomannans (US9339515B2), microRNA-19b (W02013020044A1), microRNA-21 inhibition (W02009106477A1) or other microRNAs (US20170298352A1). However, no filings mention the use of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) cultured under hypoxic conditions, i.e. reduced oxygen concentration.

[0006] EVs are nanometric (30-200 nm) circular structures secreted by essentially all cell types in physiological and pathological states. They contain bioactive components derived from the parent cell, surrounded by a lipid bilayer that protects them from rapid degradation (van Niel G, et al. Nat Rev Mol Cell Biol. 2018, 19:213-228). EVs are recognised as important mediators of intercellular communication and, due to their ability to carry bioactive molecules such as proteins, lipids and various RNA molecules, they can influence the phenotype and properties of other cells. Due to their functionality, biocompatibility and efficiency in delivering components to target cells, EVs are increasingly being considered as nextgeneration therapeutics for the treatment of human diseases (Herrmann IK, et al. Nat Nanotechnol. 2021, 16:748-759).

[0007] The aim of the invention is to provide an ingredient suitable for the treatment of organ fibrosis and a method of obtaining it.

[0008] Essence of the invention

[0009] The subject of the invention is a method for obtaining extracellular vesicles (EVs) derived from induced pluripotent stem cells (iPSCs) characterised in that: a) iPS cells are cultured in a known medium for the culture of induced pluripotent stem cells with the addition of an antibiotic on vitronectin-coated substrate, the culture being carried out under physiological hypoxia, preferably in an atmosphere containing 5% O2, and then b) extracellular vesicles are isolated from the medium collected from the iPSC cell culture, preferably at a cell density of 70-90%.

[0010] Preferably, in step a) the iPS cells are cultured in E8 medium.

[0011] Preferably, in step a) the iPSC culture is passaged, preferably every 4 days, with adding of a ROCK kinase inhibitor to the culture.

[0012] Preferably, in step a) the culture of the iPS cells is carried out in an atmosphere with 80-90% humidity. Preferably, in step a) the culture of the iPS cells is carried out by changing the culture medium once a day, preferably every 24 hours or so.

[0013] Preferably, in step b) extracellular vesicles are isolated using ultracentrifugation or ultrafdtration methods combined with size-exclusion chromatography.

[0014] Another subject of the invention is extracellular vesicles derived from induced pluripotent stem cells (iPS-EVs) obtained by the method of the invention defined above for use in the treatment of organ fibrosis.

[0015] Preferably, the extracellular vesicles (iPS-EVs) for use of the invention are used as a preparation in saline solution or in combination with hyaluronic hydrogel.

[0016] In the context of fibrotic disease treatment, scientific publications in recent years have indicated that EVs derived from iPS cells, but cultured under standard conditions with atmospheric oxygen concentrations, exhibit anti-fibrotic properties both in vitro and in vivo (Povero D, et al. JCI Insight. 2019, 5:el25652; Sun M, et al. Braz J Med Biol Res. 2021, 54:e9206; Zhou Y, et al. Int Immunopharmacol. 2021, 99:108075). However, none of the available articles shows the possibility of significantly enhancing the anti-fibrotic effect by modulating the culture conditions of iPSCs, an effect that is not obvious. In the studies that led to the present invention, it was unexpectedly found that EVs derived from culture under hypoxia, especially at 5% O2, more strongly inhibit pro-fibrotic gene expression in activated cardiac fibroblasts, compared to EVs derived from normoxia (21% O2) or from culture under 3% O2. These results were also confirmed in vivo, in an experimental mouse model of cardiac fibrosis. The beneficial effects of EVs obtained from hypoxia (5% O2) were also confirmed in vitro in models of lung, liver and pancreatic fibrosis (lung fibroblasts, liver and pancreatic stellate cells). Given the versatility of the drug obtained according to the invention and its efficacy in inhibiting fibrosis in various organs, its use in the treatment of human fibrotic diseases could be of real benefit.

[0017] Detailed description of the invention

[0018] In a preferred implementation of the invention, EVs with potent anti-fibrotic activity are derived from human iPSCs cultured under hypoxic conditions at 5% O2. Human iPS cells are characterised by the expression of the markers: OCT4, NANOG, SOX2, CD133, SSEA4. Under hypoxia, increased expression of the transcription factors OCT4 and NANOG occurs. hiPS-EVs isolated by ultrafiltration combined with size -exclusion chromatography show stronger anti- fibrotic properties than hiPS-EVs isolated by ultracentrifiigation.

[0019] In a preferred implementation of the invention, hiPS-EVs derived from a hypoxia condition of 5% O2 inhibit the fibrosis process in cardiac fibroblasts to a higher degree than hiPS-EVs isolated from a hypoxia condition of 3% or normoxia.

[0020] In a preferred implementation of the invention, under in vivo conditions - in an experimental mouse model of angiotensin Il-induced cardiac fibrosis, hiPS-EVs from the 5% O2 hypoxia condition reduce inflammation and markers of fibrosis (a-smooth muscle actin, type I and type III collagen) to a higher degree than hiPS-EVs derived from normoxia.

[0021] In a preferred implementation of the invention, hiPS-EVs derived from 5% O2 hypoxia have a universal effect on inhibiting fibrosis processes in different tissues (lung, liver and pancreas), as demonstrated using in vitro models.

[0022] In order to better explain the essence of the invention, it is discussed by means of the embodiments below further illustrated by the accompanying figures, whereby:

[0023] Figure 1 shows the characteristics of human induced pluripotent stem cells (hiPSCs). A. Microscopic image of hiPS cell colonies observed under visible light (BF - bright field) and immunofluorescence staining for pluripotency markers: SSEA4, OCT4 and CD133. Staining of cell nuclei - DAPI. B. Expression analysis of transcription factors associated with pluripotency: OCT4, NANOG and S0X2 by real-time PCR. Cell culture conditions: N - in the presence of atmospheric oxygen concentration (normoxia - N); H5 - in hypoxia 5% of O2; H3 - in hypoxia 3% of O2. *p<0.05; **p<0.01; ***p<0.001. Figure 2 shows the characteristics of EVs derived from iPSCs isolated by ultrafiltration combined with size-exclusion chromatography (UF+SEC) and ultracentrifiigation (UC) methods. The UF+SEC method used filter tubes with different protein cut-off levels according to the size: 10, 50 and 100 kDa, A. Exemplary plots of EV size and concentration analysis using the NanoSight instrument. B. Comparison of the size of EVs isolated with different methods (n=3). C. Image of EVs observed with transmission electron microscopy. D. Western blot analysis of proteins typical of EVs: CD9, CD81, Flotillinl, proteins from the culture medium (Transferrin), pluripotency markers (0CT4, CD 133, E-Cadherin), with reference to the control - P-tubulin. E. Analysis of the amount of protein in EVs isolated by UF+SEC or UC method, per ml of conditioned medium (CM). F. Average number of EV particles measured with the NanoSight instrument, per ml of CM. G. Transcript level for the NRF2 gene in EVs isolated with the UF-lOkDa+SEC method, compared to the UC method, measured by real-time PCR. H. Analysis of transcript levels for pro-fibrotic genes (ACTA2 and CCN2) in cardiac fibroblasts stimulated with TGF and treated with EVs isolated with the UF-lOkDa+SEC or UC method. Measured by real-time PCR. *p<0.05; **p<0.01; ***p<0.001.

[0024] Figure 3 shows the characteristics of EVs derived from iPS cells cultured under different oxygen concentration conditions in the culture: normoxia - 21% O2 (EV-N), hypoxia 5% O2 (EV-H5) and hypoxia 3% O2 (EV-H3). A. Exemplary plots of concentration and size measurements of EVs taken with the NanoSight instrument. B. Analysis of the average EV size. C. Image of EVs observed with transmission electron microscopy. D. Measurement of the number of particles in EV preparations, per ml of conditioned medium (CM). E. Western blot analysis of proteins typical of EVs: Syntenin, Flotillinl, CD9, CD81, proteins from culture medium (Transferrin), pluripotency markers (OCT4, CD133, E-Cadherin), endoplasmic reticulum protein (negative marker) in relation to control - P-tubulin. F. Analysis of CD81 protein levels in EV preparations from different oxygen concentration conditions. *p<0,05.

[0025] Figure 4 shows a comparison of the effect of EVs derived from different oxygen concentration conditions in culture, on the inhibition of cardiac fibroblast fibrosis. EVs derived from 3 different iPSC cell lines cultured under standard oxygen conditions (21% O2; EV-N) or reduced oxygen concentrationhypoxia: 5% O2 (EV-H5) or 3% O2 (EV-H3), were added to cardiac fibroblast cultures stimulated with TGF- factor (1 ng / mL). A. Analysis of the phenotypic transition of fibroblasts into myofibroblasts. B. Western blot detection of a-SMA protein levels and densitometric measurement (C). D. Analysis of expression levels of fibrosis-related genes (ACTA2, COL1A1, COL3A1), using real-time PCR. *p<0.05; **p<0.01; ***p<0.001.

[0026] Figure 5 shows the analysis of cardiac inflammation in a mouse model of fibrosis induced by Angiotensin II administered in osmotic pumps implanted subcutaneously. A. Haematoxylin-eosin staining. Inflamed areas with visible immune cell infiltration are marked with black ellipses (top panel) and magnified (bottom panel). B. Analysis of immune cell infiltration in the heart. Group designations: PBS - control mice without fibrosis induction and without administration of EVs that received buffered saline; Angl4 - mice with induced fibrosis, analysed on day 14 after osmotic pump implantation; Ang28 - mice with induced fibrosis on the day the experiment ended (28 days after osmotic pump implantation). Mice with induced fibrosis that received EVs derived from the atmospheric oxygen concentration condition - EV-N or from the 5% O2 hypoxia condition - EV -H5. **p<0.01; ***p<0.001; ****p<0.0001. Figure 6 shows the analysis of the presence of collagens in mouse hearts after fibrosis induction with Angiotensin II, administered in osmotic pumps implanted subcutaneously, and treated with EVs. A. Microscopic images of mouse hearts after staining with Sirius Red dye. Collagens are stained in red. B. Analysis of the degree of collagen content in the tissue. Results are presented as % of tissue area. Group designations: PBS - control mice without fibrosis induction and without administration of EVs, which received buffered saline; Angl4 - mice with induced fibrosis, analysed on day 14 after implantation of osmotic pumps; Ang28 - mice with induced fibrosis on the day the experiment ended (28 days after implantation of osmotic pumps). Mice with induced fibrosis that received EVs derived from the atmospheric oxygen concentration condition - EV-N or from the 5% O2 hypoxia condition - EV-H5. Figure 7 shows the analysis of the degree of fibrosis in the hearts of experimental mice treated with EVs. A. Analysis of transcript levels for key genes in the fibrosis process (Acta2 and Coll al, Col3al, Ctgf), performed by real-time PCR. B. Analysis of pro-fibrotic protein levels (a-Sma, Col lai and Col3al), performed using Western blot technique. C. Densitometric analysis of protein levels relative to control protein (Vinculin). *p<0.05; **p<0.0I.

[0027] Figure 8 shows a study of the effect of hypoxia-derived hiPS-EVs (5% O2) on the inhibition of lung fibroblast fibrosis induced by TGF-pi (1 ng / mL). Three doses of EVs were tested: 1.25 (EVI), 2.5 (EV2), 5 (EV3) x i0e4 EV particle s / cell. A. Percentage of cells with myofibroblast phenotype. B. Analysis of mRNA levels for genes involved in fibrosis, using real-time PCR. C. Western blot analysis of pro-fibrotic protein levels. Representative images of membranes are shown. D. Densitometric analysis of a-SMA and C0L1A1 protein levels. Control (CTRL) cells in the plots were non-stimulated with TGF-pi and non-treated with EVs. *p<0.05; **p<0.0I; ***p<0.00I; ****p<0.000I.

[0028] Figure 9 shows a study of the effect of hypoxia-derived hiPS-EVs (5% O2) on the inhibition of liver stellate cell fibrosis induced with TGF-pi (1 ng / mL). Three doses of EVs were tested: 1.25 (EVI), 2.5 (EV2), 5 (EV3) x i0e4 EV particle s / cell. A. Percentage of cells with myofibroblast phenotype. B. Analysis of mRNA levels for genes involved in fibrosis, using real-time PCR. C. Western blot analysis of pro-fibrotic protein levels. Representative images of membranes are shown. D. Densitometric analysis of a-SMA and C0L1A1 protein levels. Control (CTRL) cells in the plots were non-stimulated with TGF-pi and non-treated with EVs. *p<0.05; **p<0.0I.

[0029] Figure 10 shows a study of the effect of hypoxia-derived hiPS-EVs (5% O2) on the inhibition of pancreatic stellate cell fibrosis induced with TGF-pi (1 ng / mL). Three doses of EVs were tested: 1.25 (EVI), 2.5 (EV2), 5 (EV3) x 10e4 EV particles / cell. A. Percentage of cells with myofibroblast phenotype. B. Analysis of mRNA levels for genes involved in fibrosis, using real-time PCR. C. Western blot analysis of protein levels. Representative images of membranes are shown. D. Densitometric analysis of a-SMA and COL1A1 protein levels. Control (CTRL) cells in the plots were non-stimulated with TGF-p and non-treated with EVs. *p<0.05; **p<0.0I; ***p<0.00I; ****p<0.000I.

[0030] Figure 11 shows the dynamics of EV release from hyaluronic hydrogel (HA) of different cross-linking. Hyaluronic hydrogel (1% w / v) was prepared with EVs (1% v / v) and increasing concentrations of cross- linker (PEGDA): 0.1, 0.5, 1.0, 2.0 and 4.0% (w / v), either without (A) or with the addition of hyaluronidase (Hial; 0.01 mg / mL) (B). Samples were incubated at 37°C for 96 h. The concentration of released EVs was measured every 24 h using NanoSight instrument. The plots show the number of EV particles / ml at a given time point. C. Comparison of the cumulated number of EVs released from the hyaluronic hydrogel with and without hyaluronidase over 96 h, relative to the cross-linking density of the gel. D. Comparison of the EV release from HA with Hial. or without the enzyme, for the gel crosslinking conditions at which the greatest amount of EVs is released. *p<0.05; **p<0.01; ***p<0.001. Figure 12 shows the proteomic analysis of hiPS-EVs isolated from three hiPS cell lines cultured under different oxygen conditions (N - normoxia, 21% O2; H5 - hypoxia 5% O2; H3 - hypoxia 3% O2). A. Heat map of differentially expressed proteins in hiPS-EVs derived from different oxygen conditions. B. Number of proteins with differential expression in hiPS-EV (inclusion criteria: proteins in all 3 samples based on at least 2 peptides).

[0031] Figure 13 shows the differential expression analysis of proteins detected in hiPS-EVs derived from different oxygen conditions by liquid chromatography-mass spectrometry (LC-MS / MS). A. Volcano plots of differentially expressed proteins compared in pairs. B-D. Pathway analysis for differentially expressed proteins in each EV comparison pair (B. EV-H5 vs. EV-N; C. EV-H3 vs. EV-H5; D. EV-H3 vs. EV-N) using the ShinyGO tool, based on the Reactome database.

[0032] Figure 14 shows the identification of the most abundant proteins in hiPS-EV based on the LC-MS / MS analysis. The 5% most abundant proteins in hiPS-EVs from three hiPSC lines grown under different oxygen conditions (normoxia - 21% O2 - EV-N; hypoxia 5% O2 - EV-H5; hypoxia 3% O2 - EV-H3) were subjected to functional enrichment analysis using the STRING tool, based on WIKIPathways: EV-N (B), EV-H5 (C) and EV-H3 (D). E. Venn diagram showing the relationship between the pathways identified in hiPS-EV under each oxygen condition tested and the protein network identified for the NRF2 pathway.

[0033] Figure 15 shows Western blot analysis of antioxidant proteins from the NRF2 pathway in hiPS-EVs derived from different oxygen conditions (normoxia - 21% O2 - EV-N; hypoxia 5% O2 - EV-H5; hypoxia 3% O2 - EV-H3). A. Representative membranes. B. Densitometric measurement of antioxidant protein levels in hiPS-EV, n=6 (PRDX6) or n=9 (GSTP1; PRDX1; HSP90B). C. Pooled analysis of levels of proteins associated with the NRF2 signalling pathway in hiPS-EV (PRDX6, GSTP1, PRDX1), n=24.

[0034] Example 1. Effect of oxygen concentration conditions in iPS cell culture on the expression levels of transcription factors regulating pluripotency. iPS cell culture under different oxygen conditions.

[0035] Three iPS cell lines were used in the experiments (Gibco; a cell line from Prof. Toni Cathomen - University of Freiburg - Medical Center, Frieburg, Germany; and a previously published cell line (Bobis-Wozowicz S, et al. Stem Cells. 2015, 33:2748-2761). The iPS cells were cultured in Essentials medium (Gibco / Thermo Fisher Scientific), supplemented with antibiotic (Penicillin / Streptomycin; Gibco) on substrate coated with human recombinant vitronectin (Gibco).

[0036] E8 medium is a chemically defined, albumin-free medium developed by the laboratory of James Thomson [Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8:424-9] designed for the culture of induced pluripotent stem cells (iPSCs) for both clinical and research applications. E8 medium is a chemically defined medium containing Eagle's medium as modified by Dulbecco (DMEM) / F12 [Dulbecco, R. and Freeman, G. (1959) Virology 8:396], 64 mg / L magnesium L-ascorbic acid 2- phosphate, 14 pg / L sodium selenite, 100 pg / L fibroblast growth factor (FGF)2, 19.4 mg / L insulin, 543 mg / L NaHCCF and 10.7 mg / L transferrin, 2 pg / L transforming growth factor (TGF)[31 or 100 pg / L Nodal. The osmolarity of the medium was adjusted to 340 mOsm at pH 7.4.

[0037] The culture was maintained in an atmosphere containing different oxygen concentrations: 21% (normoxia; N) - in a standard culture incubator or at reduced oxygen levels (hypoxia; H) - 5% O2 (label: H5) or 3% O2 (label: H3). Hypoxic conditions were achieved in an Inviv02 chamber (Ruskinn). Cells were passaged every 4 days, detached from the medium with 50 pM EDTA solution (Gibco), then sedimented by centrifugation at 190 g and seeded into new vitronectin-coated culture dishes at a ratio of 1:6-8, adding 10 pM ROCK kinase inhibitor (Y -27632; Millipore) to the culture for 24 h. The cultures were maintained in an atmosphere of 5% CO2, 80-90% humidity, changing the medium every 24 hours or so.

[0038] Cells cultured under different oxygen concentration conditions were analysed for the presence of markers typical ofiPS cells: OCT4, SSEA4, CD133, using immunofluorescence staining. Forthis, cells were fixed with a 4% paraformaldehyde solution for 15 min at room temperature. Then, cells were washed with distilled water and permeabilised with 0.1% Triton-X-100 for 15 min at room temperature. After another wash with PBS solution (2x), the cells were blocked in PBS with 2% FBS for 20 min. In the next step, the cells were incubated overnight with primary antibodies (1:200 dilutions) at 4°C. A list of the antibodies used is provided in Table 1. Then, the cells were washed with PBS solution (2x) and incubated with the respective secondary antibodies for one hour at room temperature. The final step was again to wash the cells twice with PBS solution. Cell nuclei were stained with DAPI (4',6-diamidino-2- phenylindole; Invitrogen) solution. Cells were imaged with Leica DMI6000B version AF7000 fluorescence microscope.

[0039] Table 1 Summary of antibodies used in the experiments.

[0040] The level of transcripts for selected transcription factors typical for pluripotent cells: 0CT4, S0X2, NANOG was also examined. For this purpose, total RNA was isolated from cells using commercially- available GeneMATRIX Universal RNA / miRNA Purification Kit (EURx). RNA concentration and purity were determined using an Implen spectrophotometer. The reverse transcription reaction was performed using 1-2 pg of RNA and NG dART RT-PCRKit (EURx) in a C 1000 Touch Thermal Cycler (BIO-RAD). The procedure was performed according to the instructions provided by the manufacturer. The synthesized cDNA was used to analyse the expression level of selected genes by real-time quantitative PCR (qPCR), using SybrGreen dye (Applied Biosystems / Thermo Fisher Scientific) and primers of sequences presented in Table 2. The reaction was carried out in 7500 Fast Real-Time PCR System thermal cycler (Applied Biosystems), under the following conditions: 50°C - 2 minutes; 95°C - 10 minutes; and 40 cycles: 95°C - 15 seconds; 60°C - 1 minute. Based on the obtained cycle values in which the signal from the sample exceeded the established cycle threshold (Ct) for the tested gene in relation to the reference gene P-2 -microglobulin ( 2m) (ACt), the expression values of the analysed genes were calculated ( 2-AACt). Table 2. List of primer sequences used for real-time PCR.

[0041] Results:

[0042] As indicated by the obtained results, iPS cells showed the presence of marker proteins: 0CT4, SSEA4 and CD133, in all oxygen conditions (Fig. 1A). Under hypoxic conditions (5%, 3% O2), cells showed increased transcript levels for a key pluripotency gene (OCT4) comparing to normoxia (Fig. IB), and also increased mRNA levels for the NANOG transcription factor under 5% O2 hypoxic conditions, with respect to the remaining conditions tested (Fig. IB).

[0043] Conclusions:

[0044] The obtained results prove that changing the oxygen concentration conditions in iPS cell culture results in a change in the expression of transcription factors regulating pluripotency (OCT4, NANOG). The highest mRNA levels for the OCT4 and NANOG genes were obtained in the condition of culture with 5% O2. Example 2. Isolation of EVs from iPSCs

[0045] EVs were isolated from the conditioned medium collected from the iPS cell cultures, at a cell density of 70-90%, using ultracentrifugation (UC) or ultrafdtration (UF) combined with size-exclusion chromatography (SEC). The media were collected and frozen at -80°C until isolation. After thawing the media (overnight, at 4-16°C), the media were centrifuged sequentially at 4°C at increasing speeds: 500 g, 8 min (to remove dead cells and cell debris) and 2000 g, 15 min (to remove apoptotic bodies). Then, using the ultracentrifugation method, EVs were pelleted at a speed of 100,000 g, at 4°C for an hour. The EV pellet was washed with PBS solution and centrifuged again under the same conditions. The obtained EV preparations were suspended in PBS solution and frozen at -80°C. Using the UF+SEC method, the media were concentrated in Amicon concentrating tubes (Merck), with a capacity of 15 mb and a cutoff level of protein size of 10, 50 and 100 kDa. After filling the tubes, the media were centrifuged at a speed of 1800 g for approx. 40-60 min at 4°C. After the media had been concentrated approximately 50-60 times, they were transferred to low -adhesion 1.5-mL tubes and kept on ice. Concentrated preparations were purified from proteins, lipids and other small molecular substances with sizeexclusion chromatography method using an EV isolation column (70qEV, Izon). The column was washed with PBS solution filtered through 0.22 pm pore size filters. The fraction containing EVs (1.3 mb in volume) was collected in a 1.5 mb low-adhesion tube after removing the empty fraction (3 mb). EV preparations were further concentrated using 4 mb concentrating tubes (Amicon) with a protein size cut-off of 10 kDa. Finally, EV preparations were obtained, suspended in PBS solution and concentrated approximately 300 times compared to the initial volume. EV preparations were aliquoted and stored at -80°C.

[0046] The size of EVs was analyzed using a NanoSight instrument (Malvern). Samples were diluted 1: 1000 in PBS to obtain the optimal particle density for measurement. Data was collected at camera level 11 and detection threshold 5. A 60-second video was recorded for each sample. Three preparations were analyzed for each type of EVs.

[0047] The resulting vesicles were imaged using a transmission electron microscope (TEM). Negative staining of EVs was achieved by adsorption of 20 pL of EVs suspended in PBS onto nickel-coated grids (Agar Scientific, Stansted, UK) for 30 min, followed by fixation for 5 min in 2.5% glutaraldehyde solution. After removing excess liquid with filter paper, the samples were stained with 2% uranyl acetate for 30 min, washed three times with distilled water for 1 min, and dried. Then, EVs were imaged using a JEOL JEM2100 HT CRYO LaB6 transmission electron microscope (JEOL, Peabody, MA, USA).

[0048] In order to analyze the presence of selected proteins by Western blot, EVs were lysed using RIPA lysis buffer (1 / 5 volume) with the addition of protease and phosphatase inhibitors. The lysate was centrifuged for 10 min at 4°C at a speed of 10,000 g and transferred to fresh 1.5-mL tubes. The amount of protein in the preparation was measured spectrophotometrically using a kit based on the reaction with bis- cinchonic acid - BCA (Invitrogen / Thermo Fisher Scientific), according to the manufacturer's instructions. Then, loading buffer was added to each sample in a 1:3 ratio followed by sample denaturation at 95°C for 5 min. The samples were separated by mass using electrophoresis in a 12% polyacrylamide gel. The separation process was carried out at a constant voltage of 100 V for approximately 70 minutes. After this time, the separated proteins were transferred to a PVDF membrane (BioRad) using Trans-Blot Turbo transfer kit (BioRad) at a constant current of 1.3 A and a voltage of 25 V for 7-10 minutes. Protein immunodetection was performed on appropriately cut membranes relative to protein mass. The membranes were blocked in 3% BSA solution in TBST buffer for 1 h at room temperature. In a further step, the membranes were incubated overnight at 4°C with primary antibody solutions diluted 1: 1000 or 1:2000 in a 1% BSA solution in TBST. The next day, the membranes were incubated with a solution of HRP -conjugated secondary antibodies (1:2000-6000 dilution in 1% BSA in TBST) for 50 minutes. After this time, the membranes were washed 3 times in TBST buffer for 10 min. Signal detection for individual proteins was performed in a ChemiDoc XRS+ chemiluminescence instrument (Bio-Rad), after 5-minute incubation with HRP substrate (Crescendo; Merck). The list of antibodies used is presented in Table 1. Densitometric analyzes of the obtained bands were performed using Quantity One software (Bio-Rad).

[0049] Analysis of the abundance of selected transcripts in EVs was performed by real-time PCR as described above, except that RNA was isolated using Total Exosome RNA and Protein Isolation Kit (Invitrogen / Thermo Fisher Scientific).

[0050] The obtained EV preparations were tested in an in vitro cardiac fibrosis model using human cardiac fibroblasts (hCF, Cell Applications / Merck, #306-05A), which were cultured in Advanced DMEM / F12 medium supplemented with 10% FBS and P / S mixture antibiotics, under standard conditions: temperature 37°C, 95% humidity, 5% CO2. Fibrosis experiments were performed in medium containing 2% FBS, supplemented with TGF-pi (Coming; working concentration 1 ng / mL), which was added for 6 h to induce fibrosis. Then, EVs were added to the cells at a concentration of 2.5 x 10e4 EV particle s / cell for 24 h in the presence of TGF-pi (1 ng / mL). After this time, cells were harvested for analysis of the levels of selected transcripts using real-time PCR as described above.

[0051] Results:

[0052] The UF+ SEC method and sequential ultracentrifiigation were used for EV isolation. According to the latest reports from the literature, isolation based on UF and SEC allows for obtaining EVs with higher biological functionality than the UC method (Mol EA, et al. Nanomedicine. 2017, 13:2061-2065), which was important due to the subsequent use of EVs for in vitro and in vivo research. Membranes of different pore sizes were used for ultrafiltration, allowing for the concentration of proteins with a molecular weight of up to 10, 50 and 100 kDa, to compare the efficiency of EV isolation. In accordance with the guidelines of the International Society for Extracellular Vesicles (ISEV; Thery C, et al. J Extracell Vesicles. 2018, 7:1535750), the obtained EVs were characterized using various research methods. EVs isolated using different methods: UC and UF+ SEC using fdter tubes of different levels of protein size cut-off (10, 50, 100 kDa) did not differ significantly in size (diameter is approximately 210 nm), as measured by NanoSight instrument (Fig. 2A,B). In the electron microscope, EVs were visible as circular nanostructures, similarly in all preparations (Fig. 2C). EVs showed the presence of surface markers typical of exosomes (CD9, CD81) and microvesicles (Flotillin 1 ), as well as the presence of pluripotency- related proteins (OCT4, CD133, E-Cadherin), regardless of the isolation method (Fig. 2D). In terms of the amount of protein obtained per 1 mb of conditioned medium, the isolation based on UF+SEC using filtration tubes with a protein size cut-off level of 10 kDa (UF-lOkDa+SEC) turned out to be the most efficient (Fig. 2E). Using this method, on average, more than 4 times more protein was obtained than using the UC method or filter tubes with a protein size cut-off level of 100 kDa, and almost 2 times more than using 50 kDa ultrafiltration tubes. Similarly, the UF-lOkDa+SEC method led to the highest number of EV particles per 1 mb of conditioned medium (Fig. 2F). Moreover, analysis of the transcript level for NRF2, a key factor involved in the antioxidant response in cells, important in inhibiting fibrosis, showed its enrichment in EVs isolated by the UF-lOkDa+SEC method, comparing to the UC method (Fig. 2G). In an in vitro fibrosis model using TGFp-stimulated cardiac fibroblasts, the addition of EVs isolated with UF-lOkDa+SEC resulted in a 66% reduction in ACTA2 transcript levels, a key marker of activated myofibroblasts (Fig. 2H, top panel). The use of EVs obtained by the UC method, however, resulted in a reduction of the ACTA2 transcript level to 45%. Moreover, EVs obtained by the UF- lOkDa+SEC method inhibited the expression of the CTGF gene, which is a mediator of the fibrosis process, by 60%, while EVs obtained by the UC method by 47% (Fig. 2H, lower panel).

[0053] Taking into account the higher anti -fibrotic activity of EVs isolated using the UF-lOkDa+SEC method compared to EVs isolated using the UC method, the former method was selected for further analyses. In this way, preparations of EVs derived from iPSCs cultured in various oxygen concentration conditions were prepared: 21% O2, i.e. atmospheric oxygen concentration (EV-N; normoxia) and physiological hypoxia- 5% and 3% O2 (EV-H5; EV-H3) , i.e. conditions characteristic ofthe pluripotent cell niche. Additionally, EVs preparations derived from biologically neutral cells - dermal fibroblasts (DF) - were prepared and used as a control. The obtained EVs were characterized in terms of size using NanoSight instrument (Fig. 3A). It was shown that EVs originating from hypoxia have a smaller diameter compared to EVs originating from normoxia (average values: 226±15 nm for EV-N; 217±16 nm for EV-H5 and 200±15 nm for EV-H3; Fig. 3B). In the image obtained with TEM, EVs originating from different oxygen concentration conditions showed similar shapes (Fig. 3C). However, in terms of the number of EVs particles per 1 mb of conditioned medium, the largest number of them was obtained in the H5 hypoxia condition (Fig. 3D). EVs from all tested oxygen conditions showed the presence of markers typical for EVs (CD81, CD9, Syntenin, Flotillin-1), factors associated with pluripotency (OCT4, CD133, E-Cadherin), transferrin and did not show the presence of calnexin (Fig. 3E). In the case of CD81 protein, its level was significantly higher for EV-H5 relative to EVs-N (Fig. 3F).

[0054] Conclusions: EV isolation based on UF+SEC using filtration tubes with a protein size cut-off level of 10 kDa turned out to be the most efficient in terms of the amount of protein obtained and the number of EV particles per 1 mL of conditioned medium. Moreover, EVs obtained with this method were enriched in the mRNA for the antioxidant gene NRF2, compared to EVs obtained with the UC method. The use of EVs obtained with the UF-lOkDa+SEC method to inhibit fibrosis of cardiac fibroblasts significantly reduced the ACTA2 and CCN2 transcript levels to a higher extent than EVs isolated with the UC method.

[0055] Isolation of EVs from iPSCs cultured in different oxygen concentration conditions (21, 3 and 5% O2) led to obtaining EVs with similar characteristics, but different in size - EVs with a smaller diameter were obtained in hypoxic conditions compared to normoxia. EVs derived from the H5 condition were enriched for the CD 81 marker, relative to EV-N.

[0056] Example 3. Antifibrotic activity of EVs originating from different oxygen conditions in an in vitro fibrosis model

[0057] EVs derived from iPSCs cultured under different oxygen concentration conditions (EV-N, EV-H5 and EV-H3), as well as EVs derived from dermal fibroblasts (EV-DF), were tested in an in vitro cardiac fibrosis model as described above. The same amount of EVs was used: 2.5 x 10e4 EV particles / cell. RNA analyzes were performed after 24 hours from the addition of EVs, and protein analyzes by Western blot and immunofluorescence staining were performed 5 days after the addition of EVs, changing the culture medium containing TGFp (1 ng / mL) every day.

[0058] To determine the percentage of myofibroblasts, immunofluorescence staining was performed according to the protocol described in Example 1, detecting a-SMA protein.

[0059] Gene expression analysis was performed by real-time PCR according to the procedure described in Example 1, using the primer pairs included in Table 2. GAPDH and 18SrRNA were used as reference genes.

[0060] The levels of selected proteins were measured by Western blotting as described in Example 2, using the specific antibodies listed in Table 1.

[0061] Results:

[0062] The effectiveness of EVs originating from various oxygen conditions in activated cardiac fibroblasts was evaluated. Analysis of the efficiency of phenotypic transitions revealed that under the influence of TGF-pi, hCFs undergo a phenotypic transition acquiring properties characteristic of myofibroblasts (Fig. 3A). In the control condition, a small percentage of cells with clearly marked stress fibers made of a-SMA were obtained, indicating the myofibroblastic phenotype of the tested cell population(approx. 20%). The number of myofibroblasts increased significantly under the influence of TGF-pi (up to approx. 60%). The addition of control EVs isolated from human dermal fibroblast populations did not reduce the percentage of myofibroblasts and thus the efficiency of phenotypic transitions in hCF populations stimulated by TGF-pi. Similarly, no significant reduction in the number of myofibroblasts was obtained after the addition of EVs derived from normoxia and hypoxia of 3% O2. Only after the application of EVs derived from hypoxia of 5% O2, a significant inhibition of the phenotypic transition from fibroblasts to myofibroblasts was obtained, to a level similar to the control (Fig. 4A). These observations were also confirmed by analyses of a-SMA protein expression (Western blot) Fig. 4B,C. Moreover, a significant decrease in gene expression for ACTA2, collagen I and III was demonstrated (Fig. 4D). These results confirmed the observed phenomenon of the strongest inhibition of FMT by EVs isolated from media collected from iPSC cultures grown in hypoxic conditions of 5% O2.

[0063] Conclusions:

[0064] EVs derived from iPSCs cultured in the presence of 5% O2 effectively inhibit the phenotypic transition of fibroblasts to myofibroblasts, compared to EVs isolated from iPSCs under other oxygen conditions (21% O2 or 3% O2) and EVs isolated from dermal fibroblasts. The reduced number of myofibroblasts was accompanied by a decrease in the expression of genes related to fibrosis, i.e. ACTA2, COL1A1, COL3A1, to the highest extent for EV-H5, compared to other EVs tested.

[0065] Example 4. Inhibition of the fibrosis process in vivo, in a mouse model of cardiac fibrosis

[0066] Induction of fibrosis in mice (8-week-old males of the NOD / SCID strain) was performed using angiotensin II (Ang II; Sigma-Aldrich), which was administered subcutaneously using osmotic pumps (Alzet, Cupertino model 1004) at a dose of 1.4 mg / kg / day, according to a previously published protocol (Cambier L, et al., Hypertension. 2018, 72:370-380). 14 days after the induction of fibrosis, the animals received 4 doses of EVs, at intervals of 3-4 days. EVs derived from normoxia and hypoxia 5% O2 were administered. After 28 days, the experiment was ended by humanely sacrificing the mice, from which the heart was then collected and used for molecular and immunohistochemical (IHC) analyses. IHC staining was performed on paraffin sections of cardiac tissue. The heart was fixed in 10% buffered formalin solution and embedded in paraffin. Then, the tissue was cut into 4-pm-thick sections and stained with hematoxylin and eosin (H&E) for morphological analyses. The obtained preparations were histopathologically assessed for the presence of inflammation, according to the available scale: 0 = none,

[0067] 1 = infiltration of inflammatory cells around the vessels, 2 = <50% of the tissue, 3 = 50%-75% of the tissue, 4 = >75% of the tissue.

[0068] Staining for collagen was performed according to a protocol described previously (Puchtler et al., 1973; Junqueira et al., 1979). Tissue sections were stained with Sirius red dye in an aqueous solution of picric acid (Merck) for 1 hour. Then, the slides were washed twice with glacial acetic acid (0.5% v / v), then dehydrated in ethanol solutions and washed in xylene. After staining, sections were sealed with Histofluid mounting medium (Paul Marienfeld, Lauda-Konigshofen, Germany). Quantification of collagen in samples was performed using QuPath software (Bankhead et al., 2017). The results were presented as the ratio of the area covered by collagen to the total area of the heart tissue . In both stainings,

[0069] 2 preparations from each tissue were assessed and the average values were included in the calculations for each experimental group (n = 5 or 6). Transcript level analyses were performed by real-time PCR as described in Example 1, with the difference that RNA was isolated from tissues using Fenozol Plus solution (A&A Biotechnology), according to the protocol provided by the manufacturer. The primer sequences are given in Table 2. The Eef2 gene was used as an endogenous control.

[0070] The level of selected proteins was analysed by Western blotting according to the procedure described in Example 2.

[0071] Results are presented as the mean of the measurements with error bars representing the standard deviation (SD). Statistical analysis was performed based on the analysis of normality of distribution using the Shapiro-Wilk test. If the data showed a normal distribution, the ANOVA test was used together with the Tukey test. If the data did not show a normal distribution, the non-parametric Kruskal-Wallis test with Dunn's post-hoc test was used to calculate statistical significance.

[0072] Results:

[0073] Hematoxylin and eosin staining showed the presence of immune cells in preparations from hearts collected from untreated mice, with the highest degree on day 14 of the experiment (Fig. 5). After 28 days, the areas with immune cell infiltration decreased, but the difference was not statistically significant. The applied treatment, in the form of therapeutic EVs, resulted in inhibition of the infiltration of inflammatory cells, and the greatest effect was achieved for EVs derived from the 5% O2 hypoxia condition (Fig. 5B). Analyses of collagen staining with Sirius red dye demonstrated the antifibrotic properties of therapeutic EVs in the in vivo model (Fig. 6). Selected representative images show areas containing collagen fibers that are stained red (Fig. 6A). Image analysis showed a significant reduction in the area occupied by fibrotic areas in the hearts of EV -treated mice. In the case of using EVs derived from normoxia, the decrease was 25 and 16% compared to the Angl4 and Ang28 groups, respectively. However, when the EVs derived from hypoxia of 5% O2 were used, the reduction of the area occupied by collagens was 50 and 44%, respectively, in relation to the Angl4 and Ang28 groups (Fig. 6B). Moreover, the molecular analyses performed showed a significant decrease in the expression of key genes in the fibrosis process: Acta2, Collal, Col3al and Ctgf at the mRNA level (Fig. 7A). Western blot analysis confirmed a decrease in the level of profibrotic proteins, to a greater extent after the use of EV-H5 compared to EV-N (Fig. 7B,C).

[0074] Conclusions:

[0075] The treatment of cardiac fibrosis in mice, in the form of EVs preparations derived from iPS cells cultured in the presence of 5% O2, results in a significant reduction in inflammation in the tissue, as well as a reduction in collagen content and a decrease in the expression of key fibrosis markers - Acta2, Colla and Col 3a 1 at the mRNA and protein levels.

[0076] Example 5. Use of therapeutic EVs for inhibiting fibrosis of various organs in vitro

[0077] To verify the universal antifibrotic effect of EVs derived from iPSCs cultured in 5% hypoxia, other in vitro models of organ fibrosis were also used: lungs, using human lung fibroblasts (hLF Lonza, #CC- 2512), as well as pancreas and liver, using human pancreatic stellate cells (hPSCs, ScienCell, #3830) and human hepatic stellate cells (hHSCs, Innoprot, #P 10653).

[0078] The cells were cultured in dedicated media: human lung fibroblasts - in DMEM medium with high glucose supplemented with 10% FBS (v / v) with the addition of antibiotics (P / S); pancreatic stellate cells - Stellate Cell Medium (ScienCell, #5301); liver stellate cells - Stellate Cell Medium Kit (Innoprot, #P60126). All cell cultures were performed under standard conditions: temperature 37°C, 95% humidity, 5% CO2. Fibrosis experiments were carried out in media without supplements (stellate cells) or in media containing 2% FBS (fibroblasts), after prior adaptation of the cells to the changed conditions. To analyse the effectiveness of EVs, the fibrosis induction model described previously was used. The percentage of myofibroblasts in the cells was examined and the expression level of pro-fibrotic genes was measured using real-time PCR and Western blot methods, according to the procedure applied to cardiac fibroblasts. Three different concentrations of EVs (1.25 (EVI), 2.5 (EV2), 5 (EV3) x 10e4 EV particle s / cell, designated EVI, EV2 and EV3) were tested to determine the minimum effective dose that significantly reduces at least one of the fibrosis markers, at least at one test level.

[0079] Results:

[0080] In the pulmonary fibrosis model, it was shown that exposure of pulmonary fibroblasts to TGF-pi led to a significant increase in the percentage of myofibroblasts compared to the control condition (Fig. 8A). The addition of therapeutic EVs resulted in a reduction in the number of myofibroblasts observed in the TGF-pi -stimulated hLF population, most strongly at the concentration of 2.5 x 10e4 EV particles / cell (EV2) (Fig. 8A). The observed anti-fibrotic properties of therapeutic EVs were confirmed by expression analyses of the genes related to the fibrosis process: ACTA2 and COL1A1, both at the mRNA level (Fig. 8B) and at the protein level (Fig. 8C,D).

[0081] In the case of TGF-pi -induced phenotypic transitions in human hepatic stellate cell populations, a significant increase in the percentage of cells with a myofibroblast-like phenotype was observed (Fig. 9A), which correlated with an increase in FMT markers both at the transcript (Fig. 9B) and protein (Fig. 9C,D) levels. In this experimental system, a strong inhibitory effect on TGF-pi -induced hHSC phenotypic transitions was observed for therapeutic EVs administered at the highest dose, i.e. 5 x 10e4 EV particles / cell (EV3). This effect was also noted in the analysis of the expression of fibrosis markers at the mRNA (Fig. 9B) and protein (Fig. 9C,D) levels.

[0082] A similar effect was observed in pancreatic stellate cells, in which the phenotypic transition from fibroblasts to myofibroblasts was induced with TGF-pi. The strong pro-fibrotic effect of TGF-pi, leading to an increase in the percentage of cells with myofibroblast-like characteristics in the hPSC population, was abolished by therapeutic EVs administered at the highest dose, i.e. 5 x 10e4 EV particles / cell (EV3) (Fig. 10A). Analyzes of the level of fibrosis markers in hPSCs stimulated by TGF- pi showed a strong effect of TGF-pi promoting the increase in expression of a-SMA gene (Fig. 10B) and protein (Fig. 10C,D). This effect was significantly reduced by the addition of therapeutic EVs, as shown by transcript level analysis (Fig. 1OB). In the case of protein analysis, a reduction in COL1A1 levels was obtained (Fig. 10C,D).

[0083] Conclusions:

[0084] The antifibrotic properties of therapeutic hiPS-EVs derived from 5% O2 hypoxia were confirmed in several selected in vitro models of tissue fibrosis (lung fibroblasts, liver and pancreatic stellate cells). These results prove the versatility of the EV effect on fibrotic processes, leading to their inhibition. Therefore, the EVs described can be used as an antifibrotic drug in the treatment of fibrosis in various organs.

[0085] Example 6: Preparation of hiPS-EVs in hyaluronic hydrogel to obtain a preparation with an extended duration of action.

[0086] In order to study the possibility of prolonged release of EVs, for use e .g . in a living organism, hyaluronic acid (HA) hydrogel was used. Different concentrations of polyethylene glycol) diacrylate (PEGDA) cross-linker were tested in the experiment: 0.1, 0.5, 1, 2, and 4%. The concentration of hyaluronic acid (HyStem; Sigma-Aldrich) was kept constant (1%), as well as the EV concentration, which was 10 times the dose effective for inhibiting fibrosis markers in fibroblast cell lines, i.e. 2.5 x 10e5 EV particles (1%). The EV release from the hydrogel was analysed without and with the presence of the hyaluronidase enzyme (Hial.). When using Hial., 1 mg / mL hyaluronidase (Sigma-Aldrich; final concentration: 0.01 mg / mL of the hydrogel) was added to the hydrogel solution. The HA preparation procedure was performed as follows: 25 pL of PEGDA + / - Hial. were added to 100 pL of HA containing EVs. The solutions were prepared in 96-well plates, in triplicate. The plates were incubated at 37°C for 15 minutes, and then 200 pL of PBS was added. Every 24 hours, 100 pL of PBS was removed from above the hydrogel and 104 pL of PBS was added instead. The collected EVs were diluted lOx to a final volume of 1 mL using PBS. EV concentration was measured using the NanoSight instrument as described above.

[0087] Results:

[0088] A dependence of the number of released EVs on the cross-linking density of the hyaluronic hydrogel was observed. At a lower degree of cross-linking (0.1-2% PEGDA), the release of EVs occurred uniformly and inversely proportional to the PEGDA concentration (Fig. HA). In the presence of hyaluronidase, generally higher amounts of EVs released from the hydrogel were recorded. In this case, the release rate of EVs was also stable at hydrogel cross-linking values of 0.1-2% PEGDA (Fig. 11B). At a density of 4% PEGDA, the EVs release rate increased over time, which can be explained by the dependence of the release rate on the hydrogel decomposition time by hyaluronidase. To select the most favourable cross-linking parameters of the hyaluronic hydrogel, at which the most EVs are released, the cumulative values of the number of EVs collected within 96 hours of the experiment were compared. The results indicate that the presence of hyaluronidase significantly increases the number of released EVs, with hydrogel cross-linking parameters of 1-4% PEGDA (Fig. 11C). The highest number of EVs released was for the HA condition without Hial. was recorded at a cross-linking density of 0.1% PEGDA, and for the condition with Hial. - 2% PEGDA. These values were compared in a plot of EVs release versus time, confirming the benefits of using hyaluronidase for the efficient release of EVs from the hyaluronic hydrogel (Fig. HD).

[0089] Conclusions: hiPS-EVs can be effectively combined with hyaluronic hydrogel to obtain a preparation with an extended EV release time. The largest amounts of EVs are released from the HA hydrogel with 2% of PEGDA and in the presence of the hyaluronidase enzyme (0.01 mg / mL).

[0090] Example 7: Analysis of proteins in hiPS-EVs originating from different oxygen concentration conditions.

[0091] Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS / MS) according to the standard procedure (Bobis-Wozowicz et al., 2015). Data analysis was performed using the MaxQuant 2. 1.4.0 engine and Andromeda server against the SwissProt database with Homo sapiens taxonomy constraints (20,404 sequences), supplemented with the Common Protein Contamination database. LFQ intensity and iBAQ quantification (E intensity / number of theoretical peptides) were enabled and standard software settings were used, including a false discovery rate (FDR) of less than 1% for peptide and protein identification. The search parameters were as follows: enzyme - trypsin; number of missed cuts - 2; static modification - carbamidomethylation (C); dynamic modifications - oxidation (M) and acetylation (N-terminal protein). Statistical analysis was performed using Perseus 1.6.7.0 software. Differentially expressed proteins were selected for pathway enrichment analysis based on t test (p<0.05). Volcano plots were generated using the VolcaNoseR web application with p<0.05 and FC>1.3

[0061] ,

[0092] Enriched pathways were identified using the Reactome package in ShinyGO v0.77, separately for proteins with increased and decreased expression, in each contrast (H3_N; H3_H5; H5_N), with p-value < 0.1, q-value < 0.05. For the most abundant proteins in EVs (5%) under a given oxygen condition, enriched pathways were identified using STRING software, based on WIKIPathways, with the FDR threshold set to 0.05.

[0093] Additionally, selected proteins identified in the NRF2 pathway were detected with Western blot. The procedure was performed as described in Example 2. The list of antibodies used is provided in Table 3.

[0094] Table 3. Antibodies used in the analysis.

[0095] Results:

[0096] Proteomic analysis revealed the presence of differential proteins in EVs originating from different oxygen conditions, as shown in the heat map (Fig. 12A). Comparison between EV-H5 and EV-H3 revealed the highest number of differentially expressed proteins (46 and 19 of up- or down-regulated proteins) (Fig. 12B). Comparison between EV-H3 and EV-N resulted in 43 and 21 proteins with increased or decreased expression, respectively. In contrast, comparison of EV-H5 and EV-N yielded only 10 and 14 up- or down-regulated proteins, respectively (Fig. 12B).

[0097] Differential proteins in each analysed contrast are shown in volcano plots (Fig. 13A). Then, pathway enrichment analysis was performed using the ShinyGO tool using the Reactome database. Pathways for proteins with decreased expression in EV-H3 compared to EV-N include vesicular transport, mitosis, cellular response to external stimulation, protein folding, and other (Fig. 13B, left panel). Pathways that were identified for proteins with increased expression for this contrast include: translation, RNA metabolism, and mRNA splicing (Fig. 13B, right panel). In turn, for proteins with reduced expression in EV-H3, as compared to EV-N, the following were demonstrated: autophagy, mitophagy, M phase of the cell cycle, presentation of MHC II antigens, protein folding, regulation of TP53 activity and other (Fig. 13C, left panel). Proteins upregulated in EV-H3, as compared to EV-N, formed the following pathways: RNA metabolism, translation, mitosis, and protein transport (Fig. 13C, right panel). For the EV-H5 vs EV-N contrast, pathways were identified only for proteins with reduced expression in EV- H5, which involved signalling via RHO proteins, mitosis, protein transport to the Golgi apparatus and their modification, N-glycan synthesis (Fig. 13D, left panel).

[0098] Taking into account the fact that the proteins that are most abundant in EVs will have the greatest impact on the biological effect in target cells, in addition to the differential analysis, an analysis of the enrichment of signalling pathways was also performed for the 5% most abundant proteins in EVs. Using the WIKIPathways database, common pathways for proteins in EVs from all tested oxygen conditions were demonstrated, including: ribosomal proteins, the pro-angiogenic VEGF-VEGFR2 pathway, glycolysis, and gluconeogenesis (Fig. 14A-C). The pathways identified for proteins in EV-N and EV- H5 also involve regulation of the actin cytoskeleton. Importantly, the most abundant proteins in EV-H5 differed from other EV types in the NRF2-regulated antioxidant pathway (Fig. 14C,D). Proteins from this pathway included: heat shock protein 90 alpha, B 1 (HSP90AB 1 , also known as HSP90B or HSP84), PRDX1, PRDX6, GSTP1, and the transporters SLC2A1 and SLC2A3. The protein -protein interaction network generated in the STRING database showed close interaction between HSP90B, PRDX1, PRDX6 and GSTP1, which were selected for further analysis.

[0099] Using Western blotting, it was shown that the levels of all tested antioxidant proteins were increased under hypoxic conditions, with the highest amounts occurring in EV-H5 (Fig. 15A,B). In particular, PRDX1 was significantly enriched in EV-H5 (relative protein level 1.5 ± 0.6) as compared to EV-N (protein level 1 ± 0.2). EV-H3 also contained an increased amount of PRDX1 protein (relative value 1.4 ± 0.5), although the difference was not statistically significant. The total content of antioxidant proteins was significantly higher in EV-H5 compared to EV-N (Fig. 15C).

[0100] Conclusions: hiPS-EVs originating from different oxygen conditions are characterized by different protein profiles. Identified groups of differentiating proteins create signalling pathways that are different for each oxygen contrast in EVs. EV-H5 show the greatest enrichment in antioxidant proteins related to the NRF2 pathway, compared to EVs derived from EV-N and EV-H3.

Claims

Claims1. A method of obtaining extracellular vesicles (EVs) derived from induced pluripotent stem cells (iPSCs), characterized in that: a) iPSCs are cultured in a known medium for culturing induced pluripotent stem cells with the addition of an antibiotic on a substrate coated with vitronectin, and the culture is carried out in conditions of physiological hypoxia, preferably in an atmosphere containing 5% O2, and then b) extracellular vesicles are isolated from the medium collected from the iPSC culture, preferably at a cell density of 70-90%.

2. The method of claim 1, characterized in that in step a) the iPSCs are cultured in E8 medium.

3. The method of claim 1, characterized in that in step a) the iPSC culture is passaged, preferably every 4 days, with adding a ROCK kinase inhibitor to the culture.

4. The method of claim 1, characterized in that in step a) the iPSCs are cultured in an atmosphere with 80-90% humidity.

5. The method of claim 1, characterized in that in step a) the iPSCs are cultured by changing the medium once a day.

6. The method of claim 1, characterized in that in step b) extracellular vesicles are isolated using ultracentrifugation or ultrafdtration methods combined with size-exclusion chromatography.

7. Extracellular vesicles derived from induced pluripotent stem cells (iPS-EVs) obtained by the method defined in the above claims for use in the treatment of organ fibrosis.

8. Extracellular vesicles (iPS-EVs) for the use of claim 7, characterized in that they are used as a preparation in saline solution or in combination with hyaluronic hydrogel.

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