US20150017122A1 - Conditioned medium obtained from placental mesenchymal stem cells and use thereof in the therapeutic treatment of preeclampsia - Google Patents

Conditioned medium obtained from placental mesenchymal stem cells and use thereof in the therapeutic treatment of preeclampsia Download PDF

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US20150017122A1
US20150017122A1 US14/367,604 US201214367604A US2015017122A1 US 20150017122 A1 US20150017122 A1 US 20150017122A1 US 201214367604 A US201214367604 A US 201214367604A US 2015017122 A1 US2015017122 A1 US 2015017122A1
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conditioned medium
mcp
igfbp
preeclampsia
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Alessandro Rolfo
Tullia Todros
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CORION BIOTECH Srl
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    • A—HUMAN NECESSITIES
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    • 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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2053—IL-8
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly

Definitions

  • Preeclampsia is a severe pregnancy-related syndrome that affects 5-10% of all pregnant women, thus representing one of the main causes of fetal-maternal mortality and morbidity worldwide.
  • Preeclampsia is a multi-systemic disorder that manifests itself during the third trimester of pregnancy with a symptomatology characterized by maternal hypertension in previously normotensive women, proteinuria higher than 0.3 grams per day and generalized edema. Together with a compromised maternal health condition, the preeclamptic syndrome presents several risk factors for the fetus, being generally accompanied by intra-uterine fetal growth defects (intra-uterine fetal growth restriction).
  • preeclampsia has been subject of intense investigation by the clinical-scientific community during the past decade, its etiopathogenesis still remains unclear and the only effective therapeutic treatment is a timed, and often premature, delivery. Even when the newborn survives to pre-term delivery, this intervention presents several risks like pulmonary diseases, retinopathy, cerebral palsy, mental retardation, cardiovascular and metabolic diseases that could also manifest during adult age. Furthermore, even though preeclampsia resolves at delivery with placenta removal, it could determine severe maternal long term complications. Among these, it was reported a significant higher risk of chronic hypertension, diabetes mellitus, chronic kidney disease and cardiovascular diseases.
  • TNF-alpha (“Tumor necrosis factor alpha”) plays a pivotal role in the activation and propagation of the inflammatory response.
  • TNF-alpha serum and placenta expression has been found significantly increased in pregnant women affected by preeclampsia compared to women with a physiological pregnancy. Moreover, it is known that TNF-alpha is co-responsible for the reduced trophoblast invasivity typical of this pregnancy-related syndrome.
  • Placenta is a complex organ made of different tissues like the mesenchyme, that represents the most abundant placental cellular component. Recent studies have demonstrated that placental mesenchyme and amniotic membranes contain a unique cellular population with a mesenchymal-stem phenotype (Huang Y C, Yang Z M, Chen X H, Tan M Y, Wang J, Li X Q, et al. Isolation of mesenchymal stem cells from human placental decidua basalis and resistance to hypoxia and serum deprivation . Stem Cell Rev. 2009; 5(3):247-55).
  • placental mesenchymal stem cells are characterized by very elevated proliferative, differentiative and self-renewal potentials as well as by immunosuppressive and anti-inflammatory activities. Moreover, PDMSCs play a key role in the regulation of reparative and proliferative processes of neighboring cells. Raster B and colleagues reported that MSCs possess the ability to spontaneously migrate towards injured tissues and organs in order to take part in the reparative process (Raster B, Göttig S, Ludwig R J, Bistrian R, Müller S, Seifried E, et al. Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells . Blood. 2006; 108(12):3938-44).
  • mesenchymal stem cells On the basis of the unique functional plasticity and differentiation properties described above as well of the high accessibility and use of placenta per se, placental mesenchymal stem cells became subject of investigation mainly in the field of regenerative medicine. Moreover, to further support PDMSCs clinical use in this field, mesenchymal stem cells are non-immunogenic, since they do not express HLA Type II and co-stimulatory molecules (CD80, CD86, CD40) necessary to directly stimulate T lymphocytes, and they are resistant to cytotoxic T lymphocytes-mediated lysis. The distinctive immunological features of these cells suggest that they could play an important role in the maintenance of fetal-maternal tolerance.
  • HLA Type II and co-stimulatory molecules CD80, CD86, CD40
  • CM conditioned medium
  • PDMSC culture medium conditioned by PDMSC obtained from the placenta of women not affected by preeclampsia
  • CM protein secreted by PDMSCs cells that most probably all together contribute to the beneficial effects of CM which were previously described
  • the inventors subjected the CM mentioned above to proteomic analysis by using a commercial array containing antibodies capable of specifically and simultaneously recognizing several cytokines, chemokines and growth factors.
  • the data obtained from this proteomic analysis revealed the presence of several factors among which are prominent interleukin 6 (IL-6), interleukin 10 (IL-10) and monocyte chemotactic protein-1 (MCP-1).
  • IL-6 interleukin 6
  • IL-10 interleukin 10
  • MCP-1 monocyte chemotactic protein-1
  • the invention relates to a conditioned medium obtainable by culturing a placental mesenchymal stem cell derived from the placenta of a pregnant woman not affected by preeclampsia, in a liquid culture medium, the conditioned medium containing at least IL-6, IL-10 and MCP-1 factors.
  • This conditioned medium is particularly useful for the therapeutic treatment of preeclampsia.
  • the invention also relates to a method of producing the conditioned medium of the invention, the use of the conditioned medium of the invention for preparing a medicament for the therapeutic treatment of preeclampsia, and anti-inflammatory therapeutic treatment methods in patients affected by preeclampsia, everything as defined in the appended claims which form an integral part of the present description.
  • placental mesenchymal stem cell indicates a mesenchymal stem cell isolated from the placenta of a pregnant woman not affected by preeclampsia.
  • the therapeutic effectiveness of these cells is really surprising, since the conditioned medium obtained by culturing mesenchymal stem cells isolated from preeclamptic placentae does not exert any anti-inflammatory effect on pathological placental explants, contrary to what has been described for mesenchymal stem cells derived from physiological placentae.
  • Placental mesenchymal stem cells can belong to different cellular populations, depending on the placental tissue of origin.
  • human placenta has a unique structure including both fetal tissue, such as chorion (chorion leave and chorion frondosum) and amnion, as well as maternal tissues, such as decidua.
  • the conditioned medium of the invention is obtained by culturing placental mesenchymal stem cells of chorionic origin.
  • chorionic mesenchymal stem cells presents the surface antigens shown in the following table, as detected by cytofluorimetric analysis.
  • a mesenchymal stem cells derived from the amnion presenting the surface antigens described in the above-reported table, will be used,
  • the conditioned medium of the invention includes at least the factors IL-6, IL-10 and MCP-1, which are cell-secreted cytokines known per se, and which are capable of exerting a significant anti-inflammatory effect.
  • the anti-inflammatory mechanism of IL-6 involves both the inhibition of TNF-alpha production and, at the same time, the induction of secretion of Interleukin-10, a factor able to inhibit the synthesis of other inflammatory cytokines.
  • the anti-inflammatory effect of MCP-1 is mediated by a potent inhibitory action towards lymphocyte cell populations.
  • PDMSC conditioned medium analysis performed by using RayBio® Human Cytokine Antibody Array 5 kit, revealed the presence of other functional modulators secreted by placental mesenchymal stem cells, thus identifying a distinct protein expression profile.
  • the conditioned medium of the invention comprises a further cell-secreted factor selected from the group consisting of: ENA-78, GCSF, GRO, GRO-alpha, IL-6, IL-7, IL-8, MCP-1, MCP-2, MCSF, MDC, ANGIOGENIN, ONCOSTATIN m, VEGF, BDNF, BLC, CKb 8-1, EOTAXIN 2, EOTAXIN 3, FLT-3 LIGAND, FRACTALKINE, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-4, IP-10, LIF, LIGHT, MCP-4, MIF, MIP-3 alpha, NAP-2, NT-3, OSTEOPONTIN, OSTOPROTEGERIN, TGF-beta 2, TIMP-1, TIMP-2, RANTES, IGFBP-3, IL1b, IL-3, MIP-1b, PIGF, IL-1a, I309, FGF9,
  • the conditioned medium is used for the therapeutic treatment of preeclampsia.
  • an additional aspect of the invention is the conditioned medium of the present invention for use in the therapeutic treatment of preeclampsia.
  • the conditioned medium of the invention comprises, according to an embodiment, a cellular fraction which consists of the placental mesenchymal stem cells for which it was obtained.
  • the conditioned medium is free of cellular components.
  • placental mesenchymal stem cells derived from pregnant women not affected by preeclampsia, preferably of chorionic origin or as an alternative of amniotic origin, may be used for the therapeutic treatment of preeclampsia.
  • placental in the context of the present invention, indicates the derivation from physiological pregnancies not affected by preeclampsia.
  • a further aspect of the present invention is a placental mesenchymal stem cell derived from a pregnant woman not affected by preeclampsia, preferably of chorionic origin or as an alternative of amniotic origin, for use in the therapeutic treatment of preeclampsia.
  • the scope of the present invention also includes a method of preparing the conditioned medium described above, which comprises the steps of:
  • conditioned medium comprising a combination of factors secreted by said placental mesenchymal stem cells, said conditioned medium comprising at least interleukin-6 (IL-6), interleukin-10 (IL-10) and monocyte chemoattractant protein 1 (MCP-1).
  • IL-6 interleukin-6
  • IL-10 interleukin-10
  • MCP-1 monocyte chemoattractant protein 1
  • the conditioned medium obtainable by the method of the invention comprises a further cell-secreted factor which is selected from the group consisting of ENA-78, GCSF, GRO, GRO-alpha, IL-6, IL-7, IL-8, MCP-1, MCP-2, MCSF, MDC, ANGIOGENIN, ONCOSTATIN m, VEGF, BDNF, BLC, CKb 8-1, EOTAXIN 2, EOTAXIN 3, FLT-3 LIGAND, FRACTALKINE, GCP-2, GDNF, HGF, IGFBP-1, IGFBP-2, IGFBP-4, IP-10, LIF, LIGHT, MCP-4, MIF, MIP-3alpha, NAP-2, NT-3, OSTEOPONTIN, OSTOPROTEGERIN, TGF-beta 2, TIMP-1, TIMP-2, RANTES, IGFBP-3, IL1b, IL-3, MIP-1b, PIGF, IL-1a, I309, F
  • the further cell-secreted factor which is present in the conditioned medium of the invention is Interleukin-8 (IL-8), which is expressed and secreted in high amounts by the cells in the conditioned medium.
  • IL-8 Interleukin-8
  • the method of the invention also comprises step (iv) of isolating from the conditioned medium obtained in step (iii) of one or more factors secreted from the placental mesenchymal stem cells.
  • the term “basal” indicates a culture medium containing inorganic salts, amino acids and vitamins usually required to support growth of mammalian cells not having particular nutritional requirements.
  • the following liquid culture media that differ for salts and amino acids content, are mentioned: Basal Medium Eagles (BME), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Nutrient Mixture F-10 (HAM's F-10) and Nutrient Mixture F-12 (HAM's F-12).
  • BME Basal Medium Eagles
  • MEM Minimum Essential Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • HAM's F-10 Nutrient Mixture F-10
  • HAM's F-12 Nutrient Mixture F-12
  • the culture medium is not supplemented with serum, in order to avoid that the growth factors contained in the serum interfere and alter the effects produced by specific factors secreted by PDMSC.
  • placental mesenchymal stem cells are cultured for a time sufficient to allow their adhesion to the culture substrate, their multiplication and the secretion of the components which characterize the above mentioned medium and make it beneficially effective for the therapeutic treatment of preeclampsia. Therefore, placental mesenchymal stem cells are cultured for at least 3 hours, preferably for at least 12, at least 24 hours, at least 48 hours, at least 72 hours or even more.
  • the conditioned medium of the invention can be processed by filtration using filters of adequate porosity which allow to retain in suspension the cellular elements and their residues.
  • the separation of the conditioned medium from PDMSC can be achieved by centrifugation followed by cell sedimentation. Therefore, in a preferred embodiment, the step of separation of the conditioned medium from the cellular component is performed by filtration or centrifugation or a combination of both. The selection of the separation method is within the knowledge and skills of skilled in the art. Even the purification of one or more of the cell-secreted factors contained in the conditioned medium is performed by methods known per se whose selection and correct use are within the reach of those skilled in the art.
  • a further object of the invention is the use of a conditioned medium as defined above or a placental mesenchymal stem cell derived from pregnant women not affected by preeclampsia, for the preparation of a medicament for the therapeutic treatment of preeclampsia.
  • the medicament is in a pharmaceutical form suitable for systemic administration, more preferably by injection, in order to ensure its diffusion into the circulation.
  • injective systems of any type, whose selection is within the knowledge of the person skilled in the art, falls within the scope of the present invention.
  • PMSCs Placenta-Derived Mesenchymal Stem Cells Isolation
  • Placenta-derived Mesenchymal Stem Cells were isolated from placentae obtained from healthy and normotensive pregnant women with physiological pregnancy. Placental tissue collection and sampling were performed after delivery and after obtaining informed consent in accordance with the guidelines of the ethics committee of OIRM Sant'Anna—Ospedale Mauriziano of Turin. Pregnancies affected by congenital malformations, chromosomal anomalies (in structure or number), infectious diseases, diabetes, cardiovascular and metabolic syndromes were excluded.
  • Placental membranes (amnion and chorion leave) were mechanically separated from the placenta.
  • placental tissue biopsies were washed several times at room temperature by using sterile HBSS (Hank's Buffered Salt Solution, in aqueous solution) (Gibco, Invitrogen by Life Technologies), in order to completely remove blood residues.
  • sterile HBSS Hort's Buffered Salt Solution, in aqueous solution
  • Biopsies were next mechanically homogenized and processed by enzymatic digestion using 100 U/ml Collagenase I, Gibco, Invitrogen by Life Technologies), 5 ⁇ g/ml Deoxyribonuclease I (DNAse I, Invitrogen by Life Technologies) in DMEM LG (Dulbecco's Modified Minimum Essential Medium Low Glucose without L-glutamina and without Fetal Bovine Serum-FBS), at 37° C. for 3 hours in a shacking thermostated water bath.
  • DMEM LG Dulbecco's Modified Minimum Essential Medium Low Glucose without L-glutamina and without Fetal Bovine Serum-FBS
  • the resulting cell suspension was then centrifuged for 5 seconds, 540 g at 4° C. in order to remove the undigested tissue residues.
  • the supernatant was collected and filtered through Cells strainer filters with pores of 70 microns in diameter. After filtration, the solution was centrifuged for 5 minutes at 540 g, 4° C. in order to pellet the cells. The supernatant was then discarded and cells were re-suspended in sterile HBSS (30 ml for every 30 grams of original tissue).
  • Ficoll Paque Premium 1,073 (GE Healthcare Europe) was layered under the solution obtained as described above, in the proportion of 1:3 relative to the starting volume.
  • the preparation was centrifuged for 20 minutes at 540 g 20° C. and mononuclear cell ring, positioned in the middle phase of the gradient, was collected, resuspended in HBSS (50 ml for every 30 grams of original tissue) and centrifuged 10 minutes at 540 g, 20° C. in order to remove Ficoll residues.
  • Cells were maintained in culture at 37° C., 5% CO 2 . At 90% of confluence, cells were splitted by treatment with trypsin TrypLE Express (trypsin of vegetable origin without animal derivates, GMP certified, Invitrogen Life Technologies) in order to promote cell expansion.
  • TrypLE Express trypsin of vegetable origin without animal derivates, GMP certified, Invitrogen Life Technologies
  • PDMSCs differentiation was obtained by using specific induction media.
  • ⁇ -MEM supplemented with 20% FCS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 2 mM L-glutamine, 20 mM ⁇ -phosphate-glycerol, 100 nM dexamethasone and 250 ⁇ M ascorbate-2-phosphate.
  • ⁇ -MEM ⁇ -MEM supplemented with 20% FCS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 12 mM L-glutamine, 5 ⁇ g/ml insulin, 50 ⁇ M indomethacin, 1 ⁇ 10 ⁇ 6 M dexamethasone and 0.5 ⁇ M 3-isobutyl-1-methylxanthine.
  • the cultures were incubated in Chondrocyte Basal Medium supplemented with R3-IGF-1 1 mL, bFGF 2.5 ML, 0.5 mL transferrin, bovine insulin 1 M, 25 mL FBS and gentamicin/amphotericin-B 0.5 mL.
  • the medium was changed twice a week for three weeks.
  • Cellular differentiation was assessed by using appropriate colorations.
  • Osteoblast differentiation was assessed by staining with Alizarin Red S. Alizarin determines the formation of insoluble and intensely colored calcium plaques, thus allowing to highlight the bone matrix.
  • Chondrogenic differentiation was assessed by Alcian Blue staining that form salt bridges between acid mucopolysaccharides polyanions, letting glycosaminoglycans be colored of blue. Adipogenic differentiation was detected by Oil Red staining, which highlights the lipids solubilized by the solvent present in the dye solution and the red-colored fat deposits.
  • PDMSCs were plated between passages 3 to 5, time when they reached the appropriate degree of purity, as demonstrated by the absence of trophoblastic and/or haematopoietic contaminants derived from the original placental tissue. More specifically, cells were plated at a density of 1 ⁇ 10 5 cells/ml in DMEM LG without Fetal Bovine Serum (FBS) at a temperature of 37° C. and 5% CO 2 . PDMSCs were cultured for at least 3 hours to a week or more. Conditioned media were then collected at the established time points, centrifuged and/or filtered to remove contaminant cellular debris. When necessary, conditioned media obtained as just described can be preserved by freezing them at ⁇ 80° C.
  • FBS Fetal Bovine Serum
  • cytokine array analysis did not detect in the conditioned medium of the invention the following proteins, as absent or present in concentrations below the array system detection limit: GM-CSF, I-309, IL-1 alpha, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-10, IL-12 p40p70, IL-13, IL-15, IFN-gamma, MCP-3, MIG, MIP-1, RANTES, SCF, SDF-1, TARC, TGF-beta 1, TNF-alpha, TNF-beta, EGF, IGF-I, THROMBOPOIETIN, PDGF-bb, LEPTIN, EOTAXIN, FGF 4, FGF 6, FGF 7, FGF 9, IGFBP-3, IGFBP-4, IL-16, NT-4, PARC, PIGF, TGF-beta 3.
  • GM-CSF I-309, IL-1 alpha, IL-1b, IL-2, IL-3, IL-4
  • conditioned medium of the invention In order to evaluate the therapeutic efficacy of the conditioned medium of the invention, we conducted a series of studies using an in vitro model represented by placental villous explants derived from pregnancies affected by preeclampsia and fetal growth restriction. In particular, it was verified if the conditioned medium of the invention was able to reduce TNF-alpha expression levels in the above mentioned placental villous explants. TNF-alpha reduction was taken as sign of a significant anti-inflammatory activity. As previously described, several clinical and experimental evidences demonstrated that this potent pro-inflammatory cytokine is over-expressed in both the placenta and the serum of pregnant women affected by preeclampsia.
  • Placentas collection and tissue sampling were performed after delivery and after obtaining informed consent in accordance with the guidelines of the O.I.R.M. Sant′Anna and Mauriziano Hospitals ethical committee (Turin, taly).
  • the diagnosis of preeclampsia was made according to the following criteria: presence of pregnancy-induced hypertension (systolic ⁇ 140 mmHg or diastolic ⁇ 90 mmHg) and proteinuria ( ⁇ 300 mg/24 h) after the 20th weeks of gestation in previously normotensive women.
  • explants formed by a villous tree portion characterized by preserved morphology and structure and of equal weight, were excised from the placental basal plate and cultured for 12 hours in 500 ⁇ l of HAM F12 medium without FBS at 37° C. and 5% CO 2 in order to equilibrate their conditions after the delivery-induced stress. After 12 hours, the culture medium was replaced with 500 ⁇ l of conditioned medium (in 12 explants) or with 500 ⁇ l of DMEM LG medium without serum (in 12 control explants). Explant cultures were incubated under the same experimental conditions for further 48 hours.
  • RNA isolation was assessed by DNAse treatment (Sigma-Aldrich) in order to remove possible genomic DNA contaminations. RNA quality was assessed by spectrophotometric analysis at 260 nm wavelength, while its purity was determined by A260/A280 absorbance ratio at 1, 8-2.
  • cDNA complementary DNA
  • RT-PCR was performed using a random hexamers approach with the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas Life Science), following manufacturer instructions.
  • TNF-alpha gene expression levels following treatment of villous explants by the conditioned medium of the invention were assessed by Real Time PCR using TaqMan primers and probes (Applied Biosystem). In order to perform a relative quantification, Real Time PCR data were compared between treated and control groups after being normalized for 18S ribosomal subunit data, used as internal reference.

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US10758571B1 (en) 2019-04-09 2020-09-01 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
EP3846782A4 (de) * 2018-09-05 2022-07-13 Children's Medical Center Corporation Verwendung von mesenchymalen stromazellexosomen in der pränatalen therapie
CN115707772A (zh) * 2021-08-18 2023-02-21 澳门大学 一种诱导剂、巨噬细胞及其应用
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GB201501302D0 (en) 2015-01-27 2015-03-11 Ostara Biomedical Ltd Embryo implantation
GB201517523D0 (en) 2015-10-05 2015-11-18 Ostara Biomedical Ltd Methods and compositions for managing reproduction
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150216907A1 (en) * 2012-09-04 2015-08-06 Pluristem Ltd. Methods for prevention and treatment of preeclampsia
US9950014B2 (en) * 2012-09-04 2018-04-24 Pluristem Ltd. Methods for prevention and treatment of preeclampsia
US11759481B2 (en) 2014-05-18 2023-09-19 Children's Medical Center Corporation Methods and compositions relating to exosomes
US20190167724A1 (en) * 2017-12-04 2019-06-06 Caire Medical-Biotechnology International Co. Neuroprotective composition, preparation process thereof and medical uses thereof
US20190167727A1 (en) * 2017-12-04 2019-06-06 Caire Medical-Biotechnology International Co. Neuroprotective composition, preparation process thereof and medical uses thereof
EP3846782A4 (de) * 2018-09-05 2022-07-13 Children's Medical Center Corporation Verwendung von mesenchymalen stromazellexosomen in der pränatalen therapie
US10758571B1 (en) 2019-04-09 2020-09-01 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US10881693B2 (en) 2019-04-09 2021-01-05 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US11129853B2 (en) 2019-04-09 2021-09-28 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
US11654160B2 (en) 2019-04-09 2023-05-23 Combangio, Inc. Processes for making and using a mesenchymal stem cell derived secretome
CN115707772A (zh) * 2021-08-18 2023-02-21 澳门大学 一种诱导剂、巨噬细胞及其应用

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