CONDITIONED MEDIUM OBTAINED FROM PLACENTAL MESENCHYMAL STEM CELLS AND USE THEREOF IN THE THERAPEUTIC TREATMENT OF PREECLAMPSIA

Abstract

The present invention relates to a conditioned medium (CM) which is obtained by culturing, in a liquid culture medium, placental mesenchymal stem cells isolated from the placental tissue of pregnant women who not affected by preeclampsia. The conditioned medium of the present invention includes at least IL-6, IL-8 and MCP-1 proteins. The conditioned medium of the present invention is effective for the therapeutic treatment of preeclampsia.

Claims

1. A conditioned medium obtainable by culturing a placental mesenchymal stem cells from the placenta of a pregnant woman not affected by preeclampsia in a liquid culture medium, the conditioned medium comprising at least the cell-secreted factors interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1).

2. The conditioned medium according to claim 1, comprising a further cell-secreted factor selected from the group consisting of ENA-78, GCSF, GRO, GRO-alpha, IL-7, 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, FGF9, TARC, PDGF-bb, LEPTIN, TNF-a and any combination thereof.

3. The conditioned medium according to claim 1, wherein the placental stem cell has a chorionic or amniotic origin.

4. The conditioned medium according to claim 1, which is cell free.

5. A method of preparing a conditioned medium according to claim 1, comprising the steps of: (i) culturing placental mesenchymal stem cells from the placenta of a pregnant woman not affected by preeclampsia in a serum-free liquid basal culture medium for at least 3 hours; (ii) separating the cell fraction from the liquid culture medium, thereby obtaining a conditioned medium comprising a combination of factors secreted by said placental mesenchymal stem cells, said combination comprising at least interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1).

6. The method according to claim 5, wherein said conditioned medium comprises a further cell-secreted factor selected from the group consisting of ENA-78, GCSF, GRO, GRO-alpha, IL-7, 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, FGF9, TARC, PDGF-bb, LEPTIN, TNF-a and any combination thereof.

7. The method according to claim 5, wherein the culturing time of the placental mesenchymal stem cells from the placenta of a pregnant woman not affected by preeclampsia is of at least 6 hours, or at least 12 hours, or at least 48 hours, or at least 72 hours.

8. The method according to claim 5, wherein the cell fraction is separated from the liquid culture medium by filtration and/or centrifugation.

9. The method according to any of claim 5, further comprising step iv) of isolating from the conditioned medium obtained in step iii) one or more of the factors secreted by said placental mesenchymal stem cells.

10. The method according to claim 9, wherein step iv) comprises isolating the MCP-1, 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, FGF9, TARC, PDGF-bb, LEPTIN, TNF-a factor or any combination thereof.

11. A method of therapeutically treating pre-eclampsia comprising: obtaining a conditioned medium by culturing in vitro an isolated placental mesenchymal stem cell population from the placenta of a pregnant woman not affected by pre-eclampsia in a basal serum-free liquid culture medium for at least three hours, wherein the stem cell population has a chorionic or an amniotic origin, wherein the conditioned medium comprises at least the cell-secreted factors interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1), and administering the conditioned medium to a subject in need thereof.

12. The method according to claim 11, wherein the conditioned medium is in a pharmaceutical form suitable for systemic administration.

13. The method according to claim 12, wherein the pharmaceutical form is suitable for administration by injection.

14. A method of antinflammatory treatment, comprising administering to a subject in need thereof a medicament comprising the conditioned medium according to claim 1.

15. The method according to claim 14, wherein the subject is affected by preeclampsia.

Description

EXAMPLE 1

Placenta-Derived Mesenchymal Stem Cells Isolation (PDMSCs)

[0040] Placenta-derived Mesenchymal Stem Cells (PDMSCs) 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.

[0041] Placental membranes (amnion and chorion leave) were mechanically separated from the placenta.

[0042] Full-thickness tissue biopsies were excised from the placental basal plate (portion of the placenta formed by chorionic villi and adherent to the uterine wall) after mechanical removal of the decidua basalis (tissue made of maternal endometrial cells modified by the interaction with the syncitiotrophoblast).

[0043] Then, 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.

[0044] Biopsies were next mechanically homogenized and processed by enzymatic digestion using 100 U/ml Collagenase I, Gibco, Invitrogen by Life Technologies), 5 μg/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.

[0045] 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).

[0046] A volume of 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.

[0047] After centrifugation, the supernatant was discarded and the cells re-suspended in DMEM LG supplemented with 10% FBS (Gibco, Invitrogen by Life Technologies) and 0.1% Gentamicin. The cells were then plated in cell culture flasks and incubated at 37° C. and 5% CO.sub.2.

[0048] Cells were maintained in culture at 37° C., 5% CO.sub.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.

[0049] In order to isolate mesenchymal stem cells from amnion, membranes were repeatedly washed at room temperature with sterile HBSS (Hank's Buffered Salt Solution, in aqueous solution) and the amnion was mechanically separated from the chorion. The amnion was then processed by enzymatic digestion, separated and cultured as described above for mesenchymal stem cells derived from placental basal plate (chorionic portion).

EXAMPLE 2

Characterization of PDMSCs

[0050] Mesenchymal stem cells isolated from term physiological placentae (basal plate-chorionic portion) as described in Example 1, were characterized by analyzing the main surface antigenic markers typical of this cell type by cytofluorimetric assay.

[0051] The presence or absence of these antigens was evaluated by using monoclonal antibodies conjugated with fluorocromes (Myltenyi, Bologna, Italy). By fluorescence evaluation, it was demonstrated that all PDMSC cell lines were positive for the expression of surface markers CD105, CD166, CD90 and CD73 and negative for the expression of HLAII, CD34, CD133, CD20, CD326, CD31, CD45 and CD14, thus showing an appropriate mesenchymal phenotype and excluding any contamination from epithelial/trophoblast cells and haematopoietic progenitors. Moreover, the cell phenotype analysis was also conducted by performing RT-PCR experiments, that showed that all PDMSCs also express the Oct4 (Octamer-binding transcription factor 4) and NANOG (Homeobox protein NANOG) genes, typical of embryonic stem cells.

[0052] In order to evaluate PDMSCs stemness, at the third passage of culture cells were examined for their differentiation potential in three different lineage: osteoblasts, adipocytes and chondroblasts. PDMSCs differentiation was obtained by using specific induction media. For osteogenic differentiation, cell cultures were incubated in a-MEM supplemented with 20% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 20 mM β-phosphate-glycerol, 100 nM dexamethasone and 250 μM ascorbate-2-phosphate. For adipogenic differentiation, cell cultures were incubated with α-MEM supplemented with 20% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 12 mM L-glutamine, 5 μg/ml insulin, 50μM indomethacin, 1×10.sup.−6 M dexamethasone and 0.5 μM 3-isobutyl-1-methylxanthine. For chondrogenic differentiation, 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.

EXAMPLE 3

Conditioned Medium Production

[0053] In order to obtain the conditioned medium of the invention, 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.sup.5 cells/ml in DMEM LG without Fetal Bovine Serum (FBS) at a temperature of 37° C. and 5% CO.sub.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.

EXAMPLE 4

Conditioned Medium Analysis by Cytokine Array

[0054] Commercially available RayBio® Human Cytokine Antibody Array 5 was used, following manufacturer instructions, in order to investigate the cell-secreted cytokines present in the PDMSCs conditioned medium of the invention. This specific cytokine array kit allows to contemporary detect 80 different cytokines present in the same sample. Specifically, the procedure is based on antibodies spotted on a membrane and able to recognize and capture cytokines when present in the analyzed sample. In the context of this experiment, the signals generated on the array membrane at the sites of immune-complex formation were quantified by densitometric analysis using the ImageQuant software. Expression levels of the so identified cytokines were not determined as absolute values, but normalized as percentage compared to a group of standard controls included in the kit, assigning to the positive controls the value 100% and to the negative controls a value of 0%. The results of the above described experiment are shown in the following table:

TABLE-US-00002 Protein % relative to standards ENA-78  17.4% GCSF  3.8% GRO  74.4% GRO-alpha   13% IL-6 119.4% IL-7  19.4% IL-8  97.5% MCP-1  53.2% MCP-2  1.0% MCSF  4.4% MDC  5.0% ANGIOGENIN  11.5% ONCOSTATIN m  7.6% VEGF  13.3% BDNF  2.3% BLC  3.3% CKb 8-1  6.0% EOTAXIN 2  4.2% EOTAXIN 3  2.1% FLT-3 LIGAND  1.4% FRACTALKINE  6.1% GCP-2  6.2% GDNF  5.1% HGF  8.6% IGFBP-1  4.0% IGFBP-2  10.3% IGFBP-4  3.1% IP-10  1.7% LIF  1.0% LIGHT  2.0% MCP-4  20.8% MIF  4.6% MIP-3alfa  3.3% NAP-2  8.6% NT-3  11.4% OSTEOPONTIN  25.3% OSTOPROTEGERIN  12.0% TGF-beta 2  1.68% TIMP-1  23.4% TIMP-2  48.4%

[0055] Furthermore, 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.

EXAMPLE 5

Evaluation of the Therapeutic Efficacy of the Conditioned Medium Obtained from PDMSC Cultures

[0056] 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.

[0057] Cultures of 24 preeclamptic placental villous explants were treated for 48 hours with the conditioned medium obtained by culturing for 48 hours PDMSCs isolated from physiological pregnancies, as previously described in Example 3.

[0058] 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. In total, twenty-four 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 μl of HAM F12 medium without FBS at 37° C. and 5% CO.sub.2 in order to equilibrate their conditions after the delivery-induced stress. After 12 hours, the culture medium was replaced with 500 μl of conditioned medium (in 12 explants) or with 500 μl of DMEM LG medium without serum (in 12 control explants). Explant cultures were incubated under the same experimental conditions for further 48 hours. Next, treated (12) and control (12) explants were collected and processed for mRNA isolation using TRIzol reagent (Invitrogen Life Technologies) following manufacturer instructions. After isolation, messenger RNA was purified 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.

[0059] The cDNA (complementary DNA), which is necessary for the analysis of the TNF-alpha expression levels, was synthesized by RT-PCR starting from 5 μg of total RNA previously isolated. 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.

[0060] Variations of 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.

[0061] Gene expression results, represented by the histogram in FIG. 1, clearly demonstrate that the treatment performed using the conditioned medium (CM) of the invention leaded to a statistically significant reduction of TNF-alpha levels in treated preeclamptic villous explants relative to controls (C) (p=0.015).