Clinical-grade extracellular vesicles derived from umbilical cord mesenchymal stromal cells: preclinical development and first-in-human intra-articular validation as therapeutics for knee osteoarthritis | Journal of Nanobiotechnology

Non-clinical (non cGMP) sEV production and enrichment

Preclinical sEV batches were obtained as reported previously [20]. Briefly, UC-MSC from three donors in passage 5 were seeded and expanded in a maintenance medium composed of Dulbecco’s modified Eagle’s medium (DMEM), high glucose, supplemented with 1% penicillin/streptomycin solution (10,000 U/mL and 10,000 μg/mL, respectively), 1% L-glutamine (200 mM) (all from Gibco, Paisley, United Kingdom), and 5% human platelet lysate (hPL) on 10-layer Nunc^™ EasyFill^™ Cell Factory^™ systems (Thermo Fisher Scientific, Waltham, MA, United States, Cat. #140400) with a density of 6250 cells/cm². After cells reached ∼70–80% confluence, the maintenance medium was discarded, and cells were washed three times with PBS 1X before addition of the induction media (DMEM high glucose + 1% L-Glutamine) for sEV production for 48 h. The recovered medium was clarified by serial centrifugations and filtrations. After obtaining the total supernatant (SN), the sEV were centrifuged at 100,000 × g for 1 h at 4 °C, the SN was discarded, and the sEV were washed with PBS 1X. The suspension was then centrifuged at 100,000 × g for 1 h at 4 °C. The SN was again discarded, and the precipitated sEV were resuspended in the remaining PBS 1X and then preserved at − 80 °C until use. Ethical approval for the donation of umbilical cords to obtain stem cells with a therapeutic purpose was obtained from the Scientific Ethical Committee of the public agency Servicio de Salud Metropolitano Oriente (CECSSMO050612) and written informed consent was obtained from the umbilical cord donor.

sEV characterization and staining

sEV characterization for size, concentration, identity, and purity assessment was performed according to MISEV guidelines [87], following the protocols previously described by our group [20] with no changes in the antibodies or equipment used. Briefly, the size mode (nm) and concentration (particles/mL) of the sEV preparations were assessed by nanoparticle tracking analysis (NTA) using a NanoSight NS300 system (Malvern Instruments Limited, United Kingdom), considering the sample dilution in the respective sEV suspension solution (PBS or Ringer Lactate). The presence of tetraspanins CD63, CD81, and CD9 (sEV surface markers), CD44 and CD90 (MSC origin markers) (BioLegend, United States of America, Cat. #397502 and Cat. #328102, respectively) and HLA DR/DP/DQ and HLA A/B/C (major histocompatibility complex class I and class II antigens, respectively) (BD Biosciences, United States of America, Cat. #564244 and eBioscience Cat. #14-9983-82) was evaluated by flow cytometry on a FACSCanto™ II cytometer (BD Biosciences, United States of America). The acquired data were analyzed using FlowJo software (V10, BD, United States of America). The presence of Syntenin-1 (sEV endosomal origin marker), Flotillin-1 (sEV membrane marker), Calnexin (endoplasmic reticulum marker), and TOMM20 (mitochondria marker) was evaluated by western blot. The structure of sEV was evaluated by transmission electron microscopy (TEM) following a previously standardized protocol established by our group [20, 94]. Images were captured using a Talos^™ F200C G2 (Scanning) TEM (Thermo Fisher Scientific) at the Advanced Microscopy Facility UMA-UC (Pontificia Universidad Católica de Chile, Santiago, Chile). sEV staining for in vitro and in vivo tracking was performed according to a previously established protocol by our group [20, 69] using the lipophilic near-infrared fluorescent cyanine dye DiR (Biotium, United States of America, Cat. #60017) as sEV-membrane staining agent and washed using MW 3000 size-exclusion exosome spin columns (Invitrogen, United States of America, Cat. #4484449) according to the manufacturer’s instructions. The stained particles were analyzed using NTA as described previously [20].

sEV cargo characterization: miRNA profiling and proteomics

Three independent UC-MSC donors were selected for the production and enrichment of sEV. The obtained sEV were characterized by NTA to determine the size mode and particle concentration and by flow cytometry to evaluate CD63 expression as sEV marker (> 90% of positive events). A value of 4 × 10⁹ sEV particles of each UC-MSC donor was used for miRNA profiling and proteomics.

The sEV-miRNA cargo profile was determined using the services of FIRALIS S.A. (Huningue, France; www.firalis.com). Briefly, miRNA profiling was performed using HTG/EdgeSeq Whole Transcriptome Assay (WTA; 2083 miRNAs), followed by sequencing on an Illumina NextSeq 500. The data obtained were normalized before the comparative analyses. The miRNA enrichment percentages were calculated by considering the number of reads of a particular miRNA and the total number of reads in the sample.

For sEV-protein cargo identification, label-free quantification (LFQ) coupled with high-resolution mass spectrometry was performed at the Clinical Proteomic Platform of the Institute for Regenerative Medicine & Biotherapy of the University of Montpellier (Montpellier, France). For this purpose, sEV were lysed, and the proteins were reduced, alkylated, and digested with trypsin using magnetic beads. The peptides were desalted and injected into a nanoLC-Q-TOF Impact II (Bruker, United States of America). Protein identification was performed with Maxquant software (V1.6.17.0; Max Planck Institute of Biochemistry, Germany). The parameters used were the following: trypsin as digestion enzyme, 1 as the number of missed cleavages, a tolerance of 10 ppm for parent ions and 0.05 Da for MS/MS spectra, the minimum peptide size was 5 amino acids, the maximum peptide mass was 4.600 Da and a protein identification false discovery rate (FDR) was set at 2.5%. The UniProt database was used as the reference (V01/02/2021). The initial protein amount normalized to LFQ intensities for each protein before data processing was performed using the LFQ-Analyst platform. Proteins that were considered contaminants and redundant were removed. LFQ data for each protein were transformed using the log2(x) formula. The data were then normalized to a normal distribution and missing values were imputed using the BCPA (Bayesian missing value imputation) method.

In vitro biological activity of UC-MSC-sEV

Cell isolation and culture

For in vitro studies, human osteoarthritic chondrocytes (huOAC), synoviocytes, and monocytes were procured following established protocols, which were reviewed and approved by the Scientific Ethics Committee of Universidad de Los Andes (approval certificate #CEC2021077). Tissue samples were collected after obtaining written informed consent from the donors, adhering to the institutional guidelines of the Universidad de los Andes. The isolation and expansion of huOAC were performed using previously described methodologies [65]. Briefly, huOAC and synoviocytes were isolated from joint tissues of patients who underwent total knee or hip replacement surgery. The cartilage tissue for huOAC isolation was sectioned into thin slices and subjected to one hour-long digestion using a protease solution (Merck KGaA, Germany, Cat. #P5147) at 37 ºC under continuous agitation. This was followed by secondary digestion in a collagenase II solution (Sigma-Aldrich, United States of America, Cat. #C6885) for 16 h at 37 °C under constant agitation. Synoviocytes were obtained by slicing the synovial membrane into approximately 1 mm² pieces and digesting them in a collagenase I solution (Sigma-Aldrich, United States of America, Cat. #C0130) under similar conditions. After digestion, huOAC and synoviocyte samples were filtered through a 40 μm cell strainer (FALCON, United States of America, Cat. #352340) to eliminate undigested tissue. The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, United States of America, Cat. #10437028), 1% P/S, and 1% L-glut under standard culture conditions. Monocytes were derived from peripheral blood mononuclear cells obtained from healthy blood donors using Ficoll-Paque^™ PLUS (Cytiva, Sweden, Cat. #171440002) following the manufacturer’s instructions. Monocytes were isolated using the EasySep^™ Human Monocyte Isolation Kit (StemCell Technologies^™, Canada, Cat. #19359) following the manufacturer’s guidelines. Upon isolation, monocytes were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco, United States of America, Cat. #12440–053) supplemented with 10% FBS, 1% P/S, 1% L-glut, 20 mM HEPES (Gibco, United States of America, Cat. #15630080), 50 μM β-mercaptoethanol (Gibco, United States of America, Cat. #21985–023), and 1% MEM non-essential amino acids (Gibco, United States of America, Cat. #11140–050) under standard culture conditions. Additionally, macrophage colony-stimulating factor (M-CSF; 20 ng/mL) (BioTechne, R&D Systems, Cat. #216-MC) was immediately added to the culture media to induce monocyte-macrophage differentiation.

sEV internalization assays

The PKH26-stained sEV internalization assay in huOAC, synoviocytes and macrophages was performed as previously described with some modifications. Briefly, PKH26 (Sigma-Aldrich, United States of America, Cat. #PKH26GL)-stained sEV were added to the culture media of either huOAC, synoviocytes or macrophages (1 × 10⁸ particles per 200,000 cells). After 24 h, the cells were fixed with 4% PFA and permeabilized with a PBS 1X + Triton X-100 0.1% solution for 10 min on vertical agitation. huOAC and synoviocytes were stained with phalloidin-Alexa Fluor 488 at a 1:600 dilution (Invitrogen, United States of America, Cat. #A12379) and macrophages were stained with anti-CD206 at a 1:500 dilution (Biolegend, United States of America, Cat. #321110). Finally, the cells were washed three times with PBS 1X before mounting on 12 mm glass slides using Fluoroshield with DAPI (Abcam, United States of America, Cat. #ab104139). Samples were left to dry at RT for at least 30 min, after which images were taken using a confocal microscope (SP8, Leica, Germany).

DiR-stained sEV internalization assay was performed in huOAC cells according to a previously established protocol by our group [2, 20, 69], with some modifications. Briefly, 10,000/well of huOAC were seeded in 4-well plates and cultured in 300 μL/well of DMEM supplemented with 10% FBS, 1% L-glut, and 1% P/S. After 24 h, cells were washed three times with PBS 1X, and a solution of 300 μL of DMEM (supplemented with 1% L-glut) containing DiR-stained sEV (35 × 10³ particles per cell) was added per well. As an internalization control, a second 4-well replicate was cultured in parallel; however, once the sEV were added, they were incubated at 4 °C. After 16 h, cells were detached and analyzed by flow cytometry on a FACSCanto^™ II cytometer (BD Biosciences, United States of America), and the data were analyzed using FlowJo software (V10, BD, United States of America). For confocal microscopy imaging, huOAC (30,000 cells/well) were cultured on a 10 mm cover glass coated with Poly-L-Lysine in a 4-well plate. The cells were treated with 30 × 10³ DiR-stained sEV/cell. Following a 16 h incubation period, huOAC were rinsed thrice with PBS 1X and fixed at room temperature (RT) for 30 min using 4% paraformaldehyde (PFA). Subsequently, nuclei were stained with Hoechst 33342 (Sigma Aldrich, United States of America, Cat. #63493) at a 1:2,000 dilution for 15 min at RT, and the samples were then mounted on a microscopy slide using fluorescence mounting medium (Dako, United States of America, Cat. # S3023). The acquired images were examined using a confocal microscope (SP8, Leica, Germany).

sEV loading with cel-miR-39

Engineered sEV were developed using electroporation to enrich them with a synthetic miRNA derived from Caenorhabditis elegans (cel-miR-39) (Ambion, United States of America, Cat. #4464076; Assay ID: MC20682). For this, sEV (2 × 10⁹ particles) loaded with cel-miR-39 (360 nM) were resuspended in buffer containing sucrose (50 mM) (Sigma-Aldrich, United States of America, Cat. #S7903) in a total volume of 100 μL. Electroporation was performed using a single 4 mm cuvette in a Nucleofector electroporation system (Lonza, Germany, Cat. #AAF-1002B & AAF-1002X) following the ER-113 program. After electroporation, the sEV were incubated for 30 min at 37 °C for membrane stabilization. Subsequently, RNAse A (Thermo Scientific, United States of America, Cat. #EN0531) was added to a final concentration of 5 μg/mL and incubated at 37 °C for another 30 min. The treated sEV were stored at − 80 °C for at least 24 h before downstream analysis. Quantification of the miRNA loaded within the sEV involved: RNA extraction (TRIzol Reagent, Invitrogen, United States of America Cat. #15596026), reverse transcription (Applied Biosystems, United States of America, Cat. #43366596), qPCR (TaqMan, United States of America, Cat. #4440040), and miRNA cel-miR-39 TaqMan assay (Applied Biosystems, United States of America, Cat. #4427975; Assay ID: 464312_mat). The engineered sEVs’ ability to transfer miRNA cargo was assessed on huOAC, synoviocytes and monocyte-derived macrophages by culturing them for 24 h with engineered sEV loaded with cel-miR-39 and sEV loaded with a scrambled sequence (Ambion, United States of America, Cat. #4464076; Assay ID: MC20682) was used as the control (15,000 particles/cell). Following the abovementioned protocols, RNA extraction, reverse transcription, and qPCR analyses were conducted to determine cel-miRNA-39 transference to cells.

Monocyte-derived macrophage polarization assay in vitro

Monocytes isolated from three healthy donors were used in a monocyte-derived macrophage (hmMØs) differentiation assay. Monocytes were seeded on a flat-bottom 96-well plate (100,000 cells/well) and cultured under standard conditions for 6–7 days in 250 μL of MLR medium (10% FBS, 1% P/S 1%, L-glut, 20 mM HEPES, 1% Non-Essential Amino Acid solution and 50 μM β-mercaptoethanol in Iscove’s Modified Dulbecco’s medium) supplemented with 20 ng/mL M-CSF (BioTechne, United States of America, Cat. #216-MC). Half of the cell culture medium was discarded every other day, and fresh MLR/M-CSF supplemented medium was added. After six days of culture, monocytes were differentiated into macrophages and characterized by flow cytometry using fluorophore-conjugated antibodies for CD68 and CD11b detection (both from BioLegend, United States of America, Cat. #333816 and Cat. #101206, respectively). At this point, sEV treatment was started at 1 × 10⁸ sEV/well in MLR/M-CSF supplemented medium (100 μL). After 24 h, the SN was collected to study the cytokine secretion by macrophages using enzyme-linked immunosorbent assay (ELISA), and macrophages were detached to determine their polarization status by flow cytometry. The secreted Interleukin-10 (IL-10), Vascular endothelial growth factor (VEGF), Interleukin-6 (IL-6), Tumor necrosis factor-alpha (TNF-α), and Interleukin-1β (IL-1β) levels were determined by ELISA (Human DuoSet ELISA, R&D Systems, United States of America, Cat. #DY217B-05, DY293B-05, DY206-05, DY210-05, and DY201-05, respectively) following the manufacturer’s instructions. To evaluate the polarization status of hmMØs, CD68 and CD11b were used to discriminate double-positive cells. Antibodies against HLA-DR (BD Biosciences, United States of America, Cat. #564244) and CD86 (BioLegend, United States of America, Cat. #305420) were used for pro-inflammatory immunophenotyping (M1 polarization markers), and antibodies for CD206 and CD163 detection (BioLegend, United States of America, Cat. #321110 and Cat. #333606) were used for anti-inflammatory immunophenotyping (M2 polarization markers). Normalization of the median fluorescence intensities (MeFI) of each M1 and M2 marker against the MeFI values obtained in no-treatment control macrophages was used to determine the polarization status of the cells: a higher proportion of HLA-DR and CD86 MeFI’s in comparison to CD206 and CD163 MeFI’s was an indicative of pro-inflammatory M1-like polarization; on the contrary, a higher fold change of CD206 and CD163 MeFI’s in comparison to HLA-DR and CD86 was indicative of anti-inflammatory M2-like polarization. Cell viability staining (1:500, LIVE/DEAD^™ Fixable Near-IR Dead Cell Stain Kit; Invitrogen, United States of America, Cat. #L34975) was added to each sample for dead cell removal. Flow cytometry data acquisition was performed using a FACSCanto^™ II cytometer (BD Biosciences, United States of America). The acquired data were analyzed using the FlowJo software (V10, BD, United States of America).

LDH-based cytotoxicity assay

Lactate dehydrogenase (LDH) release-based sEV cytotoxicity assessment was performed in huOAC following the manufacturer’s instructions (Cytotoxicity Detection Kit^PLUSLDH; Roche, Germany, Cat. #04 744 926 001). Briefly, huOAC (2,900 cells/well) were plated on a 96-well flat-bottom cell culture plate and cultured in complete medium (100 μL/well; 10% FBS, 1% P/S 1% and L-glut in DMEM). After 24 h, the medium was replaced with fresh DMEM supplemented with 1% L-glut after three washes with PBS 1X, and sEV were added according to the following doses: dose 1 = 100 × 10⁶ sEV/well; dose 2 = 400 × 10⁶ sEV/well. After 24 h, SN was recovered to quantify LDH following manufacturer’s instructions. The negative control corresponded to untreated huOAC, whereas the positive control corresponded to Triton X-100 treated huOAC.

Chondroprotective activity study

To evaluate the chondroprotective potential of sEV, menadione was used as a cell death-triggering agent in huOAC. For this purpose, huOAC (100,000 cells/well) were seeded in DMEM supplemented with 10% FBS, 1% P/S, 1% L-glut (300 μL/well) in a 24-well plate. Once 80% confluence was reached, 20 μM of menadione (Sigma-Aldrich, United States of America, Cat. #M5750-25G), and 1 × 10⁸ sEV/well were added to fresh media (300 μL). Control wells without menadione, or menadione + sEV were also considered. After 6 h, PBS 1X containing 2% FBS (300 μL) was added to each well to wash and stop the menadione effect. Immediately, the SN was recovered and reserved, and the adherent cells were dissociated using TrypLE Express Enzyme (150 μL/well; Gibco, United Kingdom, Cat. #12605093). Next, PBS 1X containing 2% FBS (150 μL/well) was again added to recover the cells that may have remained attached to the wells. SN was centrifuged at 500 × g for 5 min at 4 °C to obtain a cellular pellet for further evaluation of apoptosis using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, United States of America, Cat. #556547) according to the manufacturer’s instructions. For this, a mix of Annexin V (1.25 μL) and Propidium Iodide (2.5 μL) prepared in 50 μL of Annexin V binding buffer was added per sample and incubated in darkness for 15 min at RT. Unstained, single-stained, and dead cells were used as controls. Flow cytometry data acquisition was performed using a FACSCanto^™ II cytometer (BD Biosciences, United States of America), and the data were analyzed using FlowJo software (V10, BD, United States of America).

Evaluation of the therapeutic potential of sEV in vivo

Animals

All mice studies were performed at the Cells for Cells S.A. animal facility following protocols revised and approved by the Institutional Animal Care and Use Committee (IACUC) of the Universidad de los Andes (approval certificate #CEC2021077). C57BL/6j mice (The Jackson Laboratory; Cat. #000664) were bred and maintained in the same facility. Experiments were carried out using male and female mice aged 8 to 12 weeks, which were randomly assigned to groups according to the experimental design. Mice were housed in cages with ad libitum access to food and water, along with pleated paper and paper cones for environmental enrichment. To ensure animal welfare, a supervision protocol was implemented based on established guidelines [56]. This study complies with the ARRIVE Guidelines for reporting animal research, and a complete ARRIVE checklist is provided in Supplementary Checklist 1. No data was excluded in the analysis of the in vivo studies.

Collagenase-induced OA (CIOA) animal model

The collagenase-induced OA (CIOA) model was used as previously described [50, 81]. Three groups were established to be compared: (1) Sham (healthy control, no OA induction), (2) OA (CIOA) and (3) sEV (CIOA animals treated with sEV). For OA and sEV groups, one unit of type VII collagenase from Clostridium histolyticum (Sigma-Aldrich, United States of America, Cat. #C2399) in 5 μL PBS 1X was IA administered to the knee joints of C57BL/6j mice on days 0 and 2. Additionally, for the sEV group, on days 7 and 14, the mice of sEV group were subjected to IA injections with 2 × 10⁸ sEV diluted in 5 μL PBS 1X. On day 42, the mice were euthanized, and paws were carefully dissected to remove soft tissues, followed by fixation in 4% PFA (Merck KGaA, Germany, Cat. #1004965000).

Micro-computed tomography of X-ray (μCT) and histological evaluations

Paw samples were analyzed in a μCT SkyScan 1278 (Bruker, United States of America) using the following parameters: 0.5 mm aluminum filter, 59 ± 4 kV, 500 μA, 0.5° rotation and 360° angular range. The specimens were scanned in all spatial planes to obtain 2D and 3D digitalized images using the Nrecon reconstruction software (V1.7.4.2; Bruker, United States of America). Then, standardized regions of interest (ROI) were obtained using the DATAVIEWER analyzer software (V1.5.6.2; Bruker, United States of America). The 2D (bone mineral density) and 3D (surface-to-volume ratio) bone changes in four knee zones (medial femur, lateral femur, medial tibia, and lateral tibia of each paw) were quantified usin the CTan software (V1.18.4.1; Bruker, United States of America). Subsequently, the paws were decalcified through a two-week incubation in a 5% formic acid solution (prepared in distilled water; Merck KGaA, Germany, Cat. #100264) and embedded in paraffin for histological analysis. As previously described, tibias were sectioned frontally and stained with safranin O and Fast Green, as described for staining proteoglycans/cartilage and bone, respectively [76]. Cartilage degradation was quantified using a modified Pritzker/Osteoarthritis Research Society International (OARSI) score, as previously described [63, 81].

Immunogenic studies

For immunogenic analysis, three groups of mice were established: (1) sham, (2) OA and (3) sEV, as previously mentioned. Mice were IA injected with 5 μL PBS 1X-solution containing ∼2 × 10⁸ particles of UC-MSC-sEV. On day 10 (3 days after IA sEV administration), the popliteal lymph nodes were recovered and mechanically disaggregated. Then, cells were passed through a 40 μm filter (Falcon, United States of America, Cat. #352340) and centrifuged at 1680 rpm for 6 min and cultured for 4 h with Phorbol 12-Myristate 13-Acetate (PMA, 50 ng/mL; Sigma-Aldrich, United States of America, Cat. #P8139) and ionomycin (1 µg/mL; Sigma-Aldrich, Cat. #I0634) in the presence of 10 μg/mL brefeldin A (Sigma-Aldrich, United States of America, Cat. #B6542). Subsequently, surface staining was performed using specific antibodies against CD4 (BioLegend, United States of America, Cat. #100422) and CD25 (Biolegend, United States of America, Cat. #102012), followed by fixation and permeabilization using Cytofix/Cytoperm^™ (eBioscience, United States of America, Cat. #5523). Finally, intracellular staining for IFN-γ (BD Pharmingen, United States of America, Cat. #554411), IL-17 (BD Pharmingen, United States of America, Cat. #560666) and Foxp3 (eBioscience, United States of America, Cat. #125773–82) was achieved. Final acquisition was performed with a FACSCanto^™ II cytometer (BD Biosciences, United States of America), and the data were analyzed using FlowJo software (V10, BD, United States of America).

sEV biodistribution study

To evaluate the in vivo biodistribution pattern of sEV therapeutics, mice were IA injected with 5 μL-PBS 1X solution containing ∼2 × 10⁸ particles of freshly purified DiR-stained sEV and non-stained sEV (auto-fluorescence control) (n = 3 per group). At different time points after injection (0, 24, and 48 h), sEV fluorescence intensities were assessed using a LI-COR Odyssey imaging system (LI-COR Biosciences, United States of America) for the entire animal and excised organs according to the manufacturer’s instructions. As control of the sEV staining procedure, DiR was diluted in 100 μL PBS 1X (at a concentration of 71 μM) and then washed using size-exclusion spin columns.

In silico studies

A comparative analysis of miRNAs within the sEV derived from UC-MSC donors was performed. To ensure analytical robustness, a threshold was implemented, wherein miRNAs with a count per million (CPM) greater than 0.5 in at least two out of three samples were considered. The identified miRNAs were subsequently cross-referenced with miRNet and HMDD databases [11, 15]. The target genes associated with the identified sEV-miRNAs were selected and subjected to Gene Ontology enrichment analysis, focusing on biological processes, using the R package GOStats, GOchord, and networkD3 [18].

Concurrently, for protein analysis, proteins present in sEV with at least 2500 LFQ (protein abundance value) in at least two out of the three samples analyzed were considered. The resultant proteins were subjected to Gene Ontology enrichment analysis, specifically focusing on biological processes, using the R package GOStats, GOchord, and networkD3.

To determine the effect of sEV on putative target genes, hmMØs were subjected to the polarization assay described before and RNA was extracted (TRIzol Reagent, Invitrogen, United States of America Cat. #15596026) for reverse transcription assay. Trancript levels of STAT1 were determined by TaqMan assay (Applied Biosystems, United States of America, Cat. #4453320; Assay ID: Hs01013996_m1) and transcript levels of PPARγ were determined by qPCR using Brilliant II SYBR Green (Agilent Technologies; Cat. #600828) and the following primers: forward 5′-CCTTGCAGTGGGGATGTCT-3′; reverse 5′-CTCGCCTTTGCTTTGGTCA-3′). Both qPCR were performed in an AriaMx Real-time PCR System (Agilent Technologies).

Formulation and stability studies in sEV-based product development

Formulation evaluations

For product development and process validation studies, three sEV batches were generated and isolated at a smaller scale using a previously described protocol, except for the utilization of either PBS 1X or Ringer Lactate (RL; Baxter, United States of America, Cat. #HRB2323) during the sEV washing step and the final sEV resuspension. Each formulation was evaluated according to the following parameters: particle’s size mode (nm), concentration (particles/mL), identity markers (CD63, CD81, and CD9), and potency assays (via the hmMØs polarization assay), utilizing established protocols, as previously described. The stability assessment of the sEV-based therapeutics was conducted at 5 and 24 months after storage at − 80 °C. This evaluation encompassed sEV batches produced and enriched on a reduced scale by employing RL as the vehicle for formulation.

Stability studies

Short-term stability of sEV products after thawing was conducted at 2–8 °C in previously − 80 °C-stored sEV, which were thawed and maintained at 2–8 °C for 24 h. Both studies employed the same parameters as those previously described for evaluation.

Manufacture, quality controls and characterization of clinical grade UC-MSC-sEV

A flowchart of cells and sEV production for clinical use is illustrated in Supplementary Fig. 1.

Production of clinical grade UC-MSC

All tissue samples were obtained using protocols that were reviewed and approved by the Scientific Ethics Committee of the Universidad de Los Andes (approval certificate #CEC201861). Clinical grade cell manufacturing was carried out as previously described by our group with some modifications [30, 92348]. In brief, UC were obtained from full-term human placentas by cesarean section after signed informed consent from healthy donors following the United States of America (USA) Code of Federal Regulations (CFR) Food and Drug Administration (FDA) Title 21, Part 1271: Human Cells, Tissues, and Cellular and Tissue-Based Products, Subpart C: Donor Eligibility (§1271.45–1271.90). UC-MSC treatments were manufactured in a facility that complies with GMP in compliance with USA CFR FDA Title 21, part 1271, Subpart D: Current Good Tissue Practice (§1271.145–1271.320) and with International Organization for Standardization (ISO) certification for the Quality Management System (ISO Standard No. 9001:2015) of the UC-MSC production process at Cells for Cells S.A., Santiago, Chile (www.c4c.cl). All sterility controls were negative to approve the subsequent use of UC-MSC.

UC-MSC were cryopreserved in the third passage (p = 3) until their approval as the master cell bank (MCB) and were subsequently used in the clinic. Cell culture was performed as previously described [20, 30, 9, 48]. The UC-MSC were characterized according to the guidelines of the International Society for Cell and Gene Therapy [16]. Immunophenotyping of UC-MSC was performed using a Human MSC Analysis Kit (BD Stemflow^™, United Sates of America, Cat. #562245), and dead cells were discarded using Zombie Aqua Dye (BioLegend, United States of America, Cat. #77143). The analysis was performed by flow cytometry using a FACSCanto^™ II cytometer. The acquired data were analyzed using the FlowJo software V10. This analysis was performed using MCB cells to approve the lot for clinical use. The trilineage differentiation capacity of cultured UC-MSC was evaluated using the StemPro™ differentiation kits (Gibco, Life Technologies Corp., United States of America) following the manufacturer’s instructions: Adipogenesis Kit (Cat. #A1007001), Chondrogenesis Kit (Cat. #A1007101) and Osteogenesis kit (Cat. #A1007201). After 21 days, cell differentiation into adipocytes was confirmed by Oil Red O staining of lipidic vacuoles (Sigma-Aldrich, United States of America, Cat. #O0625) and osteocyte differentiation was confirmed by calcium deposits detected using Alizarin Red staining (Sigma-Aldrich, United States of America, Cat. #A3757). Chondrogenic differentiation was confirmed after 10 days by Safranin O staining (Sigma-Aldrich, United States of America, Cat. #S2255). Tumorigenic tests of UC-MSC in immunocompromised mice were performed under specific pathogen-free conditions at the Cells for Cells S.A. animal facility. After 3 months, organs were collected (skin, liver, lung, brain, and kidney), and histopathological analysis was performed. The previously described results showed the absence of tumors. The genomic stability of the UC-MSC over time was tested by karyotype analysis of cells at p-5 according to the USA CFR FDA Title 21, Part 211: Current Good Manufacturing Practice for Finished Pharmaceuticals, Section 110: Sampling and testing of in-process materials and drug products (§210.110). Thus, a Batch Approval Certificate (BAC) is generated, as shown in Supplementary Fig. 2.

Clinical grade sEV production and quality controls

UC-MSC approved for clinical use were thawed and seeded in maintenance medium composed of DMEM (Corning, Mediatech Inc., United States of America, Cat. #15-018-CV), supplemented with 1% P/S, 2 mmol/L L-glut and 5% fibrinogen-depleted hPL (manufactured by Cells for Cells S.A., under GMP compliance standard) [20], on a Nunc^™ TripleFlask^™ at a density of 2000 cells/cm². After the cells reached confluence, they were expanded and seeded on a 10-layer Nunc^™ EasyFill^™ Cell Factory^™ system. After the cells reached approximately 80% confluence, the maintenance medium was discarded. Cells were washed with PBS 1X (Gibco, Life Technologies Corporation, United States of America, Cat. #10010072) before the addition of the induction medium for sEV production: DMEM supplemented with only 2 mmol/L L-glut. After 2 and 4 days, SN was collected and subjected to serial centrifugation to remove cellular debris and sequential filtrations in 0.45 and 0.22 μm pore-size membrane filtration units (Thermo Fisher Scientific, United States of America, Cat. #569-0020 and Cat. #566-0020, respectively), and then, subjected to serial ultracentrifugation (Thermo Electron LED GmbH, model Sorvall WX +) at 100,000 × g for 70 min at 4 °C. The pellet obtained was washed once with RL, the supernatant was discarded and sEV were resuspended in the remaining RL, aliquoted, and stored at − 80 °C until use. The sterility of sEV was evaluated using the same protocol described above for the UC-MSC sterility assessment. Each final product of the sEV-based therapeutic for clinical use complied with standardized procedures based on GMP and all quality controls mentioned above.

First-in-human application of cGMP-sEV therapy for OA: sterility assessment, patient recruitment, and imaging protocols

For the clinical case, the selection of the clinical exploration dose was not established using the conventional allometric scaling method for human-equivalent dose (HED) typically used for small-molecule drugs. This, due to the limitations inherent to the complex manufacturing of an sEV-based product that imposes a limit on the dose of the product that can be produced, thereby restricting the range of doses feasible to administer in a clinical experience. Instead, the IA dose extrapolation for the HED was based on an evaluation of the observed preclinical and clinical efficacy of previous studies performed by our group using the sEV parental cells [47, 48], as well as an assessment of the acceptable safety risk, by the FDA’s statement “If available, previous clinical experience with the cellular and gene therapy (CGT) product or related products, even if by a different route of administration or for a different condition, may help to justify the clinical starting dose,” from the “Considerations for the Design of Early-Phase Clinical Trials of Cellular and Gene Therapy Products” guidance [17]. The exploratory dose was calculated based on our published clinical results of IA use of 2 × 10⁶ UC-MSCs in knee OA [47] (NCT No. 03810521) and our own findings indicating a secretion rate of ~ 5.3 × 10¹⁰ particles sEV of the same number of cells [20]. Considering the manufacturing feasibility of the sEV-based product, we estimate that a dose of 2 × 10¹⁰ ± 0.5 × 10¹⁰ total sEV would be required for local administration to the knee.

The cGMP-sEV (2 × 10¹⁰ ± 0.5 × 10¹⁰ particles) packed into a syringe was kept at 4 °C until IA administration in the patient. The final number of particles was determined using NTA as described above. As a UC-MSC-sEV therapeutic sterility assessment, 11 × 10⁷ particles were used for contamination determination by Aerobic/anaerobic automated blood culture system, and 14 × 10⁷ particles were used for endotoxin determination as described previously. Both control groups were negative for release of the final product. A Certificate of Analysis (CoA) was delivered, indicating that cGMP-sEV-based therapy has the necessary sEV characteristics for release and the minimum sterility requirements for patient administration (Supplementary Fig. 3).

The patient was recruited in October 2021 at the Osteoarthritis Center at the Clínica Universidad de los Andes in Santiago, Chile. Approval was obtained from the Scientific Ethical Committee of the public agency Servicio de Salud Metropolitano Oriente (CECSSMO030821). Written informed consent was obtained from the patient. Subject met the following inclusion criteria: age between 30 and 75 years, symptomatic knee OA (defined as daily pain at the affected joint for at least 3 months before inclusion and a visual analog scale equal to or greater than 40 mm), and grade II to III Kellgren-Lawrence radiographic changes. None of the following conditions was retained: meniscal rupture, bilateral symptomatic knee OA, disease of the hip and/or spine, local or systemic infection, any form of secondary arthritis, or previous malignancy. The injection was performed by an orthopedic surgeon at the superior lateral aspect of the patella using a 21-gauge, 1-inch needle. No local anesthetic was used before the puncture. Clinical outcomes (VAS and WOMAC indexes) were evaluated at 3, 6, and 12 months of follow-up.

Regarding the imaging procedure, MRI at baseline and 6 months later was analyzed by a blinded radiologist. The patient was studied using a Philips Achieva 3 Tesla MRI scanner, with Smart Knee software to achieve equal knee positioning in pre- and post-treatment resonance imaging. The MRI protocol aims to study articular cartilage volumetry to evaluate the positive changes with treatment and the absence of structural damage to the cartilage. The DICOM files were anonymized and sent electronically to a third party via a secure platform (Image Analysis Group—IAG—, London, UK) for analysis. The external company utilized proprietary software following ISO13485 and the USA CFR FDA Title 21, Part 11: Electronic Records; Electronic Signatures (§11.1–11.300), to perform Quality Controls on, segment, and quantify all MRI images. The images were analyzed by an IAG radiologist and reported to our group.

Phase I clinical trial design

The clinical investigation will represent a phase I trial focusing on UC-MSC-sEV in patients with symptomatic Kellgren II-III knee OA. The phase I component of the study will be an open-label dose escalation pilot study (NCT No. 06431152; title: “Administration of sEV derived from UC-MSC in patients with osteoarthritis of the knee: safety determination in a pilot dose-escalation study”) in which three cohorts of subjects with OA will receive increasing doses of UC-MSC-sEV administered as a single IA injection. Each cohort will comprise four participants. Specifically, patients within the cohorts will be administered the following exploration doses: 2 × 10⁹ particles/3 mL RL ± 0.5 × 10⁹ particles (first cohort-low dose), 6 × 10⁹ particles/3 mL RL ± 0.5 × 10⁹ particles (second cohort-median dose), or 2 × 10¹⁰ particles/3 mL RL ± 0.5 × 10⁹ particles (third cohort-high dose). Eligible study subjects will be enrolled at the Clínica Universidad de los Andes. The selection of the study subjects will be performed following the inclusion and exclusion criteria shown in Fig. 8E.

The UC-MSC-sEV will be prepared at the Cells for Cells S.A. GMP facility based at the Clínica Universidad de los Andes. The sEV-based therapeutic for clinical use will be manufactured in compliance with standardized procedures based on GMP regulations and all quality controls aforementioned. The sEV therapeutic will be transported to the patient administration site under controlled conditions, ensuring maintenance of a temperature range between 2–8 °C. The sEV injection is expected to be administered within the first 6 h of product manufacture.

The primary study endpoints of this trial will focus on the safety, feasibility, and toxicity of the sEV-based product. The phase I will examine: (1) the incidence of immediate post-infiltration adverse reactions in patients; (2) the occurrence of synovitis post-infiltration in patients at 24 and 48 h, as well as on days 7 and 15; (3) the frequency of post-infiltration pain reported by patients at 24 and 48 h, and on days 7 and 15; and (4) the prevalence of adverse events related to sEV therapy occurring beyond IA infiltration at 24 and 48 h, and on days 7 and 15, as well as at months 2, 4, 6, 8, 10, and 12. The secondary study endpoint will be determine the optimal dose for phase II trials. The criteria that will be considered are: (1) Safety profile at infiltration at 24 and 48 h, and on days 7 and 15, as well as at months 2, 4, 6, 8, 10, and 12; (2) changes in WOMAC scores at months 2, 4, 6, 8, 10, and 12; and (3) alterations in the VAS pain scores at months 2, 4, 6, 8, 10, and 12.

Statistical analysis

All figure legends include n involved. Analyses were performed using GraphPad Prism (V10.2.0; United States of America). For the data normality test, a Shapiro–Wilk test was performed, followed by one-way ANOVA with Tukey’s multiple comparisons test or unpaired Student’s t-test, depending on the number of groups to be evaluated. For large datasets, outliers were removed using the robust regression and outlier removal (ROUT) method (Q = 1%). For non-parametric data, a Kruskal–Wallis test followed by Dunn’s multiple comparison post-test was performed. For in vivo model, there were no criteria set for including/excluding animals. P-values < 0.05 were considered significant in all cases (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant).