WO2021067421A1 - Préparation de cellules souches mésenchymateuses humaines thérapeutiquement améliorées et leurs utilisations - Google Patents

Préparation de cellules souches mésenchymateuses humaines thérapeutiquement améliorées et leurs utilisations Download PDF

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WO2021067421A1
WO2021067421A1 PCT/US2020/053521 US2020053521W WO2021067421A1 WO 2021067421 A1 WO2021067421 A1 WO 2021067421A1 US 2020053521 W US2020053521 W US 2020053521W WO 2021067421 A1 WO2021067421 A1 WO 2021067421A1
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hmscs
ifp
msc
hpl
mscs
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Diego CORREA
Dimitrios KOUROUPIS
Annie C. BOWLES
Lee D. Kaplan
Thomas M. BEST
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University Of Miami
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/98—Xeno-free medium and culture conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/115—Platelets, megakaryocytes

Definitions

  • the present disclosure generally relates to methods for preparing human mesenchymal stem cells (hMSCs) that express high CD 10 phenotypes.
  • the present disclosure further relates to methods of treating local inflammation, fibrosis, and/or musculoskeletal pain using hMSCs that express high CD 10 phenotypes.
  • MSC Mesenchymal Stem Cell
  • This invention provides methods for producing clinically relevant amounts of MSCs that do not involve FBS or other animal-derived media, supplements, or components, wherein the MSCs produced thereby can be more safely used for treating appropriate diseases and disorders in humans and other animals.
  • hMSCs human mesenchymal stem cells
  • hMSCs human mesenchymal stem cells
  • Also provided herein is a method of treating musculoskeletal pain in a subject in need thereof comprising administering to the subject an effective amount of human mesenchymal stem cells (hMSCs).
  • hMSCs human mesenchymal stem cells
  • Also provided herein is a method of repairing altered blood vessels impacting their capacity of responding with changes in vessel tone and to contribute to inflammation and fibrosis comprising administering to the subject an effective amount of human mesenchymal stem cells (hMSCs).
  • hMSCs human mesenchymal stem cells
  • FIGS 1A-1C illustrate that IFP-MSCs showed high growth kinetics and clonogenicity when expanded in either hPL or Ch-R.
  • Figure 1A and Figure IB demonstrate that IFP-MSCs expanded in either hPL or Ch-R showed a higher growth rate until confluency as compared with FBS alone.
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • MSC mesenchymal stem cell.
  • Figures 2A-2B illustrate that IFP-MSCs expanded in either hPL or Ch-R have an enhanced immunophenotypic profile.
  • Figure 2A shows surface markers assessed in noninduced IFP-MSCs show MSC-defining markers highly expressed in all culture conditions, whereas CD 10 and CD 146 (immunomodulatory) and CXCR4 (migratory) showed increased expression only in hPL and Ch-R.
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • MSC mesenchymal stem cell.
  • Figures 3A-3B show steogenic, chondrogenic, and adipogenic differentiation potential of IFP -MSC expanded in either hPL or Ch-R.
  • Figure 3 A shows that hPL- and Ch-R- expanded IFP-MSCs showed superior qualitative differentiation capacity upon induction in vitro for bone (mineral deposition assessed by alizarin red staining), fat (lipid accumulation assessed by oil red staining), and cartilage (glycosaminoglycan production assessed by toluidine blue staining) as compared to FBS-grown cells.
  • Figure 3B illustrates that quantitative molecular profiling showed that differentiation-related markers in cultures expanded in hPL and Ch-R were increased versus those expanded in FBS, indicating their mature status.
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • H&E hematoxylin and eosin
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • MSC mesenchymal stem cell.
  • Figures 4A-4B illustrate that IFP-MSCs expanded in either hPL or Ch-R had a reduced inflammatory baseline signature while strongly secreting reparative growth factors.
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • MSC mesenchymal stem cell
  • TIC TNF ⁇ , ⁇ F ⁇ , and CTGF.
  • FIG. 5 illustrates FP-MSCs expanded in either hPL or Ch-R showed high growth factor secretion when compared with FBS plus priming.
  • Ch-R chemically reinforced medium; FBS, fetal bovine serum; hPL, human platelet lysate; IFP, infrapatellar fat pad; MSC, mesenchymal stem cell.
  • Figures 6A-6C illustrate IFP-MSCs expanded in either hPL or Ch-R effectively degraded substance P (SP) in vitro.
  • Figure 6A illustrates endogenous levels of SP (quantified at ⁇ 250 pg/mL and shown as dotted colored lines) and exogenously added recombinant SP (quantified at ⁇ 1100 pg/mL and shown as a gray dotted line) were used for reference (as bottom and top boundaries, respectively) to quantify SP degradation activity by the cells and their supernatant in the different media formulations.
  • FIG. 6B illustrates CD10 immunolocalization was detected in IFP-MSCs as a concentrated punctate signal (red) around cells and highly present in hPL- and Ch-R-expanded IFP-MSCs (blue nuclei).
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • MSC mesenchymal stem cell.
  • Figure 7 illustrates IFP-MSCs expanded in either hPL or Ch-R effectively reduced synovitis and IFP fibrosis in vivo.
  • Hematoxylin and eosin (H&E) staining top 2 panels
  • Masson trichrome staining middle panel
  • substance P immunolocalization lower 2 panels
  • SP1 fibers in areas of active inflammation and fibrosis showed dramatic reduction for the synovium and the body of the IFP in groups similarly correlated with the degree of CD10 positivity. Of note, almost no signal was detected in healthy control rats, supporting the high specificity of the SP signal. Arrows in the top panel indicate areas of synovitis.
  • Ch-R chemically reinforced medium
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • IFP infrapatellar fat pad
  • IHC immunohistochemistry
  • MIA monoiodoacetate
  • MSC mesenchymal stem cell
  • SP substance P.
  • Figure 8 illustrates CD10 expression levels in crude and CD10 immunomagnetically sorted non-induced and TIC-primed IFP-MSC.
  • Figure 9 illustrates the results of real time quantitative PCR (RT qPCR) showing expression of inflammatory cytokine with relative fold changes calculated using the FBS-grown data as reference (set up as 1). Compared to non-induced hPL, FBS, and Ch-R IFP-MSC.
  • RT qPCR real time quantitative PCR
  • Figure 10 illustrates protein association network of non-induced and TIC-primed hPL and Ch-R expanded IFP-MSC.
  • STRING analysis of the proteins with statistical differences between hPL/Ch-R and FBS in naive or TIC- priming conditions (total of 41) was performed using all available interaction sources and 0.4 as a confidence interaction score.
  • Figures 11A- 11B illustrate protein interactomes of non-induced and TIC-primed hPL and Ch-R expanded IFP-MSC.
  • Figure 11A and 11B show biological processes and KEGG/reactome pathways analyses revealed different type and number of proteins affected in naive and TIC-primed IFPMSC.
  • Figures 12A-12C show CD 10 immunophenotype in IFP-MSC and BMMSC pre- and post- ⁇ and TIC priming and hPL expansion.
  • Individual donors in each hMSC type are presented with distinctive shapes and color tones to allow intra-donor comparisons between naive and both primed methods, as well as between DMEM/FBS and hPL expansion of IFP-MSC.
  • FIG. 13 illustrates CD 10 surface expression in various MSC types cultured in regulatory-complaint condition.
  • CD10 expression in various MSC types CD90 (>95%) and CD34 ( ⁇ 5%) as known markers UC-MSC (human umbilical cord - Wharton’s Jelly) BM- MSC (human bone marrow) IFP-MSC (human infrapatellar fat pad).
  • All MSC types were grown in human platelet lysate (hPL) medium which is a regulatory-complaint medium for further in vivo therapeutics.
  • All MSC types (UC, BM, IFP) at passage 3 grown with hPL medium demonstrated MSC related phenotypic profile CD34-CD90 + CD 146 + showing also variable expression levels of ACE2 and CD142 expression.
  • CD10 showed higher expression levels in UC- and IFP-MSC compared to BM-MSC.
  • Figure 14 illustrates CD 10 mechanism of action in Renin- Angiotensin system.
  • FIG. 15 illustrates that MSC-bound and -released CD 10 strongly degraded Angiotensin 1 in vitro. All MSC types tested efficiently degrade Angiotensin 1 (ANG1) via both the released and cell-bound CD 10 protein. Moreover, CD 10 expression was paralleled with the significant reduction in ANG1 levels exercised by all naive MSC populations, suggesting a CD 10-dependent ANG1 degradation. The abrogation of this effect after inhibiting the enzymatic activity of CD 10 with thiorphan (CD 10 inhibitor) strongly supports this statement. Importantly, CD 10 released from MSC can equally or in some cases stronger (UC MSC) degrade ANG1 compared to the CD10 cell-bound.
  • UC MSC UC MSC
  • FIG 16 illustrates the MSC exosomal isolation and characterization strategy disclosed in particular embodiments herein.
  • MSC exosomes were isolated by ultracentifugation and CD63-based magnetic enrichment. NanoSight analysis revealed exosomes of size ⁇ 200nm that are stained strongly positive (90 ⁇ 10%) for the exosomal surface marker CD9 indicating their purity.
  • Figure 17 illustrates that CD10 actions are mediated via both CD 10-bound exosomes and soluble CD 10 protein.
  • FBS fetal bovine serum
  • hPL human platelet lysate
  • Ch-R Chemically-reinforced medium
  • the present disclosure generally relates to methods for preparing human mesenchymal stem cells (hMSCs) that express high CD 10 phenotypes.
  • the present disclosure further relates to methods of treating local inflammation, fibrosis, and/or musculoskeletal pain using hMSCs that express high CD10 phenotypes.
  • the methods for preparing human mesenchymal stem cells described herein advantageously provide hMSCs with increased proliferative, phenotypic, differentiation, secretory and functional profiles directly related to consistently efficient Substance P (SP) degradation in vitro and in vivo, even at significantly lower cell doses.
  • SP Substance P
  • the human mesenchymal stem cells described herein allow for a significantly-reduced cell dose (1/10 th of the injected cells) for the generation of efficient therapeutic outcomes. Translated into a clinical protocol, this would reduce the required number of IFP-MSC injected into numbers that can be generated in vitro in a shorter period of time.
  • the resulting protocol would require only -2-5 x 10 6 .
  • the human mesenchymal stem cells possessing high CD 10 described herein can be used to effectively suppress inflammation and fibrosis.
  • hMSCs human mesenchymal stem cells
  • CD 10 also known as neprilysin, is a surface neutral endopeptidase expressed in multiple cells, including MSC and specifically IFP-MSCs.
  • MSC surface neutral endopeptidase expressed in multiple cells
  • IFP-MSCs specifically IFP-MSCs.
  • the anti-inflammatory effects of CD10 have long been recognized in other systems, while its presence in IFP-MSCs seems to be critical for efficient SP degradation, both in vitro and in vivo.
  • the methods disclosed herein produce MSCs that express CD10 at a level equal or greater than 30%, 40%, 50%, 60%, 70%, 80% or 90% positivity overall.
  • an MSC preparation according to the present disclosure is an MSC preparation that is 70% homogeneous (i.e., 70% positivity overall) with respect to being CD 10 positive.
  • the methods disclosed herein produce MSCs that express a high CD 10 phenotype (i.e. express CD 10 at a level equal or greater than 50%, 60%, 70%, 80% or 90% positivity overall).
  • the high phenotypic expression of CD 10 of the MSCs disclosed herein is desired trait during cell-based product manufacturing processes. Based on the correlation between CD 10 positivity and therapeutic outcome, levels ⁇ 70% or more indicate a high likelihood of an effective therapeutic outcome.
  • hMSCs that express CD 10 at a high level are obtained using flow cytometry.
  • hMSCs that express CD 10 at a level equal or greater than 70% positivity overall are obtained using flow cytometry.
  • hMSCs that express CD 10 at a level equal or greater than 80% positivity overall are obtained using flow cytometry.
  • hMSCs that express CD 10 at a level equal or greater than 90% positivity overall are obtained using flow cytometry.
  • culturing of MSCs is carried out in xeno-free medium.
  • xeno-free refers to an absence of direct or indirect exposure to nonhuman animal components.
  • the xeno-free formulations for MSC culturing comprises human platelet lysate (hPL) or chemically-reinforced media (Ch-R).
  • hPL human platelet lysate
  • Ch-R chemically-reinforced media
  • culturing hMSCs comprises culturing hMSCs at 5 % (v/v) CO2 until the hMSCs are 80% confluent; passaging the hMSCs at a 1:5 ratio to Passage 3; and detaching the hMSCs.
  • the hMSCs are cultured at 37 degrees Celsius.
  • Cell priming includes preparing cells for some specific function or lineage-specific differentiation, including but not limited to cell activation, molecular signaling, genetic or epigenetic modifications, and morphology/phenotype changes.
  • the hMSCs are exposed to a proinflammatory/profibrotic environment (i.e., priming).
  • priming comprises exposing the hMSCs to TNF ⁇ , IFN ⁇ , and/or CTGF to enhance their immunomodulatory effects.
  • the cells are primed with 15 ng/ml TNF ⁇ , 10 ng/ml IFN ⁇ , and 10 ng/ml CTGF).
  • the cells are primed for 48 hours or 72 hours. The time difference is based on an attempt to mimic the cascade of events that lead from pure inflammation (TI) to a more fibrotic response (TIC), as a sequential tissue response (synovitis followed by IFP fibrosis).
  • CD 10 is present in exosomes derived from hMSCs and/or freely released by the cells to the environment.
  • the hMSCs are derived from postnatal adipose-, infrapatellar fat pad-, postnatal bone marrow-, postnatal endometrial-derived, perinatal origin umbilical cord-, or perinatal origin placenta tissues.
  • subject is intended to include human and non-human animals, particularly mammals.
  • the methods disclosed herein relate to treating a subject for local inflammation, fibrosis and/or musculoskeletal pain.
  • inflammation and/or fibrosis is arthritis, osteoarthritis, synovitis, tendinitis/tendinopathy, other musculoskeletal inflammation, neuroinflammation, or any tissue wound and/or inflammation.
  • the musculoskeletal pain is a result of arthritis, osteoarthritis, synovitis, tendinitis/tendinopathy, other musculoskeletal inflammation, neuroinflammation, or any tissue wound and/or inflammation
  • treatment refers to therapeutic treatment.
  • Those in need of treatment include subjects having local inflammation, fibrosis and/or musculoskeletal pain.
  • the methods disclosed herein can be used to treat local inflammation, fibrosis and/or musculoskeletal pain.
  • Administration refers to providing, contacting, and/or delivering a compound or compounds by any appropriate route to achieve the desired effect.
  • Administration may include, but is not limited to, oral, sublingual, parenteral (e g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.
  • composition refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject.
  • the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of human mesenchymal stem cells.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of the human mesenchymal stem cells of the disclosure.
  • the formulations of the disclosure When used for in vivo administration, the formulations of the disclosure should be sterile.
  • the formulations of the disclosure may be sterilized by various sterilization methods, including, for example, sterile filtration or radiation.
  • the formulation is filter sterilized with a pre-sterilized 0.22-micron filter.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins (2005).
  • the formulations can be presented in unit dosage form and can be prepared by any method known in the art of pharmacy. Actual dosage levels of the active ingredients in the formulation of the present disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject (e.g. , "a therapeutically effective amount"). Dosages can also be administered via continuous infusion (such as through a pump). The administered dose may also depend on the route of administration. For example, subcutaneous administration may require a higher dosage than intravenous administration.
  • IFP tissue ⁇ 20ml was mechanically dissected and washed repeatedly with Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma), followed by enzymatic digestion using 235 U/ml Collagenase I (Worthington Industries, Columbus, OH) diluted in DPBS and 1% bovine serum albumin (Sigma) for 2 hours at 37°C with agitation.
  • DPBS Dulbecco’s Phosphate Buffered Saline
  • DMEM low glucose GlutaMAX (ThermoF isher Scientific, Waltham, MA) +10% fetal bovine serum (FBS; VWR, Radnor, PA]
  • FBS fetal bovine serum
  • hPL Chemically-defined, DMEM/ 10%FB S
  • Complete hPL medium was prepared by supplementing DMEM low glucose GlutaMAX with 10% hPL solution (PL Bioscience, Aachen, Germany) and 0.024 mg/ml xeno-free heparin (PL Bioscience, Aachen, Germany).
  • Complete chemically reinforced medium was prepared by mixing Mesenchymal Stem Cell Growth Medium 2 with supplement provided according to manufacturer’s instructions (PromoCell, Heidelberg, Germany).
  • All MSC were cultured at 37°C 5% (v/v) CO2 until 80% confluent (denoted as P0), then passaged at a 1:5 ratio until P3 detaching them with TrypLETM Select Enzyme IX (Gibco, ThermoFisher Scientific) and assessing cell viability with 0.4% (w/v) Trypan Blue (Invitrogen, Carlsbad, CA).
  • TIC inflammatory/ fibrotic cocktail 15 ng/ml TNF ⁇ , 10 ng/ml IFN ⁇ , 10 ng/ml CTGF
  • Non-induced and TIC-induced cultures were evaluated for their phenotypic profiles by flow cytometric analysis.
  • RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Frederick, MD) according to manufacturer’s instructions.
  • Total RNA (lpg) was used for reverse transcription with SuperscriptTM VILOTM cDNA synthesis kit (Invitrogen).
  • Protein array of 41 growth factors (GFs) (RayBio ® C-Series, RayBiotech, Peachtree Comers, GA) was used to determine secreted levels obtained from IFP-MSC expanded in all three culturing conditions. For each population, 1 mL of conditioned media obtained from 2 donors, was prepared and used for each assay following the manufacturer’s instructions. Data shown represent 40 sec exposure in FluorChem E chemiluminescence imaging system (ProteinSimple, San Jose, CA). Results were generated by quantifying the mean spot pixel density of each array using protein array analyzer plugin using Image J software (Fiji/ImageJ, NIH website). The signal intensities were normalized with the background whereas separate signal intensity results represent the average pixel density of two spots per protein. The signal intensity for each protein spot is proportional to the relative concentration of the antigen in the sample. Pathway analysis
  • Putative interactomes were generated by Search Tool for Retrieval of Interacting Genes/Proteins (STRING 11.0; available from: http://string-db.org) database using interaction data from experiments, databases, neighborhood in genome, gene fusions, cooccurrence across genomes, co-expression and text-mining. An interaction confidence score of 0.4 was imposed to ensure high interaction probability.
  • K-means clustering algorithm was used to organize proteins into 3 separate clusters per condition tested, discriminated by colors. Venn diagrams were used to demonstrate all possible relations between naive and/or TIC-primed IFP-MSC cultured in all three different conditions for the significantly (p ⁇ 0.05) altered proteins. Functional enrichments related to biological process, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and reactome pathways were presented in radar graphs for all conditions tested.
  • SP was then quantified in centrifuged (1500 rpm; 5 minutes) conditioned media (in technical triplicates run in duplicates within the membrane) obtained from IFP-MSC cultures: i) in baseline cultures (i.e., endogenous MSC-derived SP); ii) after exogenous addition of substance P (834 pg/ml) for 35 minutes to the cell-free supernatant (i.e., supernatant group); and iii) after addition of SP (834 pg/ml) for 35 minutes to the cells in fresh medium (i.e., cells group).
  • SP final levels were determined by subtracting measured optical densities of individual wells at 450nm and 540nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA), and converted into concentrations using the reference standard curve run with the assay, and contrasted to samples with only exogenously-added SP to the medium (i.e., no cells and no supernatant).
  • IFP-MSC groups were fixed with 4% paraformaldehyde, washed with PBS, followed by incubation with tris buffered saline (TBS; Sigma- Aldrich) containing 0.05% Triton X-100 solution (Sigma- Aldrich) for 30 minutes. Groups were then incubated with blocking solution composed of TBS with 10% normal goat serum (NGS) for 1 hour. Goat anti-human CD 10 polyclonal antibody (R&D Systems) was prepared in TBS containing 1% NGS and added to samples for 1 -hour incubation at room temperature with gentle agitation.
  • TBS tris buffered saline
  • NGS normal goat serum
  • IFP-MSC were immunomagnetically separated based on CD 10 expression and further expanded in vitro. Briefly, IFP-MSC were suspended in staining buffer containing PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA and then incubated with biotinylated anti-human CD10 (Miltenyi Biotech, Inc., Auburn, CA) at RT for 20 minutes. InvitrogenTM CELLection DynabeadsTM Biotin Binder Kit (Thermo Fisher Scientific) were used according to manufacturer’s instructions for magnet-activated cell sorting resulting in the POS and NEG subpopulations. POS and NEG BM-MSC were directly plated in culture to obtain relevant numbers for the in vivo study yielding the CD10 bright and CD10 dim IFP-MSC populations.
  • BSA bovine serum albumin
  • Acute synovial/IFP inflammation was generated by intra-articular injection of 1 mg of mono-iodoacetate (MIA) in 50 ⁇ of saline in the right knee. Briefly, under isoflurane inhalation anesthesia rat knees were flexed 90° and MIA was injected into the medial side of the joint with a 27G needle using the patellar ligament and articular line as anatomical references. Three (3) days later, a single intra-articular injection of 500,000 cells for groups: naive crude, naive CD10 + (bright and dim), and 500,000 or
  • TIC-induced IFP-MSC in 50 ⁇ l of Euro-Collins solution was performed (similar injection technique), having as control: 1) rats receiving MIA but not IFP-MSC (Only MIA group); and 2) healthy rats receiving only IFP-MSC (Only IFP-MSC group). Animals were sacrificed 4 days after IFP-MSC injection (d7 total).
  • Rat knee joints were harvested by cutting the femur and tibia/fibula 1 cm above and below the joint line, muscles were removed and joints were fixed with 10% neutral buffered formalin (Sigma- Aldrich) for 14 days at room temperature. Knee joints were decalcified, cut at sagittal plane in half, embedded in paraffin and serial 4 ⁇ m sections were obtained. Hematoxylin and Eosin (H&E) staining was performed to evaluate the structure and morphology of knee joints. Substance P and anti-human Mitochondria immunolocalization were determined by immunofluorescence and immunohistochemistry staining, respectively.
  • Sections were washed with lx PBS + 0.01% Triton X-100 and incubated for 1 hour with secondary antibody containing Alexa Fluor594 conjugated goat anti-rabbit IgG antibody (Thermo Fisher Scientific) at room temperature. Controls were incubated with secondary antibody only. All sections were rinsed with lx PBS, mounted in prolong gold antifade reagent with DAPI (Invitrogen), and microscope images were acquired using Leica DMi8 microscope with Leica X software (Leica).
  • Conditioned media from IFP-MSC groups cultured for two days in exosome-depleted media (DMEM/ 10%FB S or DMEM/ 10%hPL or chemically-reinforced) were filtered through a 0.22 ⁇ m filter to remove debris and large vesicles. Subsequently collected and differentially centrifuged for 2,000xg for 10 min, 10,000xg for 30 min, and ultracentrifuged for 120,000xg for 16hr using a sucrose gradient to increase purity. After ultracentrifugation two fractions were collected: i) the supernatant and ii) the exosomal pellet.
  • DMEM/ 10%FB S or DMEM/ 10%hPL or chemically-reinforced were filtered through a 0.22 ⁇ m filter to remove debris and large vesicles. Subsequently collected and differentially centrifuged for 2,000xg for 10 min, 10,000xg for 30 min, and ultracentrifuged for 120,000xg for 16hr using
  • the supernatant was concentrated at 4,000xg for 30 min using Amicon Ultra- 15 centrifugal filter devices (Millipore) and then stored at -20°C until further experimentation .
  • Pre-enriched exosomal pellets were washed and incubated overnight at 4°C with the Dynabeads®-based Exosome- Human CD63 Isolation/Detection Reagent (Thermo Fisher Scientific). Using a magnetic separator, exosome preparations were further purified.
  • Exosomes collected from each group were assessed for multimodal parameters for biophysical and biochemical characterization: quantity and size determination through nanoparticle tracking analysis (NTA) (NanoSight NS300, Malver). Exosomal identity profiling was complemented by staining with CD9 antibody (Invitrogen) to validate their presence in CD63 + -gated particles by flow cytometry (CytoFLEX, Beckman Coulter). Data were acquired using a Cytoflex S (Beckman Coulter, Brea, CA) and analysed using Kaluza analysis software (Beckman Coulter).
  • NTA nanoparticle tracking analysis
  • Prior in vitro assessment collected exosomes were lysed using RIP A buffer ((Thermo Fisher Scientific).
  • CD10 protein levels were quantified in centrifuged (2,000g; 10 minutes) conditioned media (run in duplicates) obtained from MSC cultures in all conditions. Levels were determined by measuring the fluorescence (450nm) of individual wells in endpoint mode (SpectraMax M5 spectrophotometer, Molecular Device
  • Angl was then quantified in centrifuged (1500 rpm; 5 minutes) conditioned media (in technical triplicates run in duplicates within the membrane) obtained from the cultures: i) in baseline cultures (i.e., endogenous cell-derived Angl); ii) after exogenous addition of Angl (2500 pg/ml) for 20 minutes to the cell-free supernatant (i.e., supernatant group); and iii) after addition of Angl (2500 pg/ml) for 20 minutes to the cells in fresh medium (i.e., cells group).
  • Angl final levels were determined by subtracting measured optical densities of individual wells at 450nm and 580nm (SpectraMax M5 spectrophotometer, Molecular Devices, San Jose, CA), and converted into concentrations using the reference standard curve run with the assay, and contrasted to samples with only exogenously-added Angl to the medium (i.e., no cells and no supernatant).
  • Example 1 IFP-MSCs Show Superior Growth Kinetics When Expanded in Either hPL or Ch-Rvs FBS Growth kinetics and clonogenic potential of IFP-MSCs expanded in either hPL or Ch- R were analyzed and compared with IFP-MSCs expanded in FBS alone. All 3 culture conditions demonstrated variable growth kinetics for 8 days. In detail, hPL-expanded IFP- MSCs showed an increased growth rate, reaching 85% confluency as compared with the FBS cultures, which had only 62% confluency on day 8. Ch-R medium showed the most potent growth rate versus the other 2 culturing conditions, entering confluency (>85%) only 4 days after seeding in vitro ( Figures 1A and IB).
  • Example 2 IFP-MSCs Expanded in Either hPL or Ch-R Have Privileged
  • the common MSC- defining markers CD44, CD73, CD90, CD105, CD 166) showed a similar expression pattern (>90% positivity), while LepR and CD56 were absent, and CD271 and CD200 had low to negative expression.
  • NG2 showed high expression (approximately 90%) only in Ch-R ( Figure 2A).
  • markers related to MSC functionality toward an immunomodulatory and antifibrotic phenotype were significantly increased in noninduced IFPMSCs by solely culturing in either hPL or Ch-R.
  • IFP-MSCs The tripotential capacity of IFP-MSCs to undergo osteogenic, chondrogenic, or adipogenic differentiation was comparable in FBS, hPL, and Ch-R.
  • qualitative assessments showed that hPL- and Ch-R-expanded IFP-MSCs deposited higher levels of minerals on the monolayer surface in osteogenesis, had higher lipid vacuole accumulation within the cytoplasm in adipogenesis, and displayed stronger cartilage-specific metachromasia for glycosaminoglycans produced in chondrogenesis as compared with FBS- expanded IFP-MSCs (Figure 3A).
  • hPL- and Ch-R- expanded IFP-MSCs showed significantly (P ⁇ .05) higher expression levels for the osteogenic gene OMD, adipogenic gene FABP4, and chondrogenic genes ACAN and COMP as compared with FBS expanded IFP-MSCs ( Figure 3B), indicating their increased maturity during the different differentiation schemes.
  • Example 4 IFP-MSCs Expanded in Either hPL or Ch-R Have a Reduced Baseline
  • IL-8 was the only cytokine with significantly (P ⁇ 05) higher expression levels in hPL- and Ch-R-expanded IFP-MSCs as compared with the FBS reference sample, with 6.8- and 8.6-fold expression levels, respectively.
  • Noninduced hPL-expanded IFP-MSCs showed overall higher secretion of GFs when compared with FBS medium ( Figure 4B).
  • IFP-MSCs expanded in hPL and Ch-R without priming secreted 29 and 24 growth factors, respectively, at significantly (P ⁇ 0.05) higher levels compared with FBS.
  • TIC priming hPL- and Ch- R- expanded IFP-MSCs showed increased secretion of various proteins.
  • the overall number of proteins secreted was reduced, as TIC priming also affected the secretion of FBS-expanded IFP-MSCs.
  • hPL and Ch-R-expanded IFP-MSCs secreted 16 and 18 GFs, respectively, at significantly (P ⁇ 05) higher amounts.
  • Simultaneous Venn diagram representation of all 4 secretory profiles (noninduced and primed hPL and noninduced and primed Ch-R), when compared with their FBS counterparts (noninduced and primed), revealed a core of 7 GFs (EGF R, HGF, IGFBP-2, M-CSF R, PDGF-AA, SCF R, YEGF) that are commonly increased in those 4 groups.
  • 13 of 16 for hPL and 14 of 18 for Ch-R were shared with the secretory profiles in their noninduced state.
  • hPL IFP-MSCs showed higher enrichment of the cytokine-cytokine receptor interaction and Jak-STAT (2 of 6) pathways, whereas Ch-R IFPMSCs showed higher enrichment of the MAPK, PI3K-Akt, Ras, and Rapl (4 of 6) pathways ( Figure 11B).
  • the Jak-STAT pathway which is highly enriched in hPL IFP-MSCs, is a principal downstream mechanism for an array of cytokines and GFs and is directly involved in cytokine-cytokine receptor interaction signaling (hsa:04060).
  • Ch-R IFPMSCs except the MAPK, PI3K-Akt, and Ras pathways, which are involved in downstream signaling upon cytokine and GF activation — the Rapl pathway, which is involved in cell adhesion, cell-cell junction formation, and cell polarity, is also highly enriched.
  • priming resulted in further-boosted protein involvement in all signaling pathways for both hPL- and Ch-R-primed IFP-MSCs.
  • Example 7 IFP-MSCs Expanded in Either hPL or Ch-R Show Increased
  • Example 8 IFP-MSCs Expanded in Either hPL or Ch-R Effectively Reverse Synovitis and IFP Fibrosis and Degrade SP In Vivo
  • a rat model of induced acute synovitis and IFP fibrosis was used to confirm in vivo the degradation of SP and to test the capacity of IFP-MSCs with different levels of CD 10 positivity to reverse synovial and IFP inflammation and fibrosis.
  • CD10 + -selected IFP-MSCs showed the strongest reduction of synovitis and IFP fibrosis and SP degradation, while a reduction in the cell dose in 1 of 10 (total of 50,000 cells) still effectively generated therapeutic efficacy, as they were high in CD 10 owing to the priming stimulation.
  • Example 9 MSC-bound and -released CD10 strongly degraded Angiotensin 1 in vitro
  • Example 10 MSC exosomal isolation and characterization strategy
  • MSC-derived exosomes were isolated by ultracentifugation and CD63-based magnetic enrichment. NanoSight analysis revealed exosomes of size ⁇ 200nm that are stained strongly positive (90 ⁇ 10%) for the exosomal surface marker CD9 indicating their purity ( Figure 16).
  • Example 11 CD10 actions are mediated via both CDlO-bound exosomes and soluble CD10 protein
  • FB S fetal bovine serum
  • hPL human platelet lysate
  • Ch-R Chemically-reinforced medium

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Abstract

La présente divulgation concerne d'une manière générale des procédés de préparation de cellules souches mésenchymateuses humaines (hMSC) qui expriment des phénotypes CD10 élevé. La présente divulgation concerne en outre des méthodes de traitement d'une inflammation locale, d'une fibrose et/ou d'une douleur musculosquelettique faisant appel aux hMSC qui expriment des phénotypes CD10 élevé. Cette invention concerne des procédés de production de quantités de de MSC pertinentes sur le plan clinique, qui ne font pas appel au FBS ou à d'autres milieux, suppléments ou composants d'origine animale, les MSC ainsi produites pouvant être utilisées de façon plus sûre pour traiter des maladies et troubles appropriés chez les êtres humains et d'autres animaux.
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* Cited by examiner, † Cited by third party
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