FOETAL MESENCHYMAL STEM CELL COMPOSITION OF ALLOGENIC ORGIN, TREATMENT METHOD AND USE THEREOF IN MASTITIS IN MILK-PRODUCING ANIMALS, INCLUDING CATTLE
20220016174 · 2022-01-20
Inventors
Cpc classification
A61K9/0041
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
International classification
A61K35/28
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to the use of regenerative medicine in livestock farming, in particular to regenerative medicine for the care of livestock, and even more particularly to regenerative medicine in the care of milk-producing animals, including cattle. Specifically, the invention concerns a foetal mesenchymal stem cell (MSC) composition of allogenic origin, a treatment method and the use thereof in mastitis in milk-producing animals, including cattle, suffering from mastitis, to immunostimulate the animal, the MSC coming from foetal bovine adipose tissue. The composition can be administered via the intramammary route, exercising an immunomodulatory effect on the inflammatory response and strengthening antimicrobial activity against mastitis-causing pathogens in milk-producing animals with mastitis, thereby becoming an alternative to replacing and/or supplementing the use of antibiotics.
Claims
1. A composition for the treatment of mastitis in milk-producing animals, comprising fetal mesenchymal stem cells (MSC) of allogenic origin, wherein the MSC are obtained from bone marrow.
2. The composition according to claim 1, wherein the composition is formulated for an intramammary route of administration.
3. A method of treating mastitis in milk-producing animals, comprising administering a composition comprising fetal mesenchymal stem cells (MSC) of allogenic origin, wherein said MSC are obtained from bone marrow.
4. The method according to claim 3, wherein the milk-producing animal is a bovine.
5. The method according to claim 3, wherein the composition is administered via an intramammary route.
6. The method according to claim 3, wherein the composition is administered in combination with an antibiotic.
7. The method according to claim 6, wherein the antibiotic is selected from cephalosporins, penicillins, streptomycins, tetracyclines or a combination thereof.
8-13. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0047] The present composition comprises a suspension of fetal bovine MSCs of allogeneic origin that can be administered intramammary; alternative or complementary treatment and use method to the use of antibiotics. The intramammary administration composition allows the distribution of these cells in the mammary tissue, exerting an immunomodulatory effect on the inflammatory response, controlling the infectious process, and therefore, shortening the recovery time in dairy cows with induced mastitis.
[0048] The present composition can be used in combination with an antibiotic selected from cephalosporins, penicillins, streptomycins, tetracyclines, or a combination thereof.
[0049] The present composition comprises fetal mesenchymal stem cells (MSCs) of allogeneic origin and serves in the treatment of mastitis in milk-producing animals, including bovines. The present composition is a substitute or complementary alternative to the use of antibiotics, increasing the effectiveness of the treatment and reducing the risk that its excessive use represents for public health.
[0050] The present regenerative mammary gland composition comprises allogeneic MSCs that are prepared by the following steps (
[0051] Cryopreservation and commercialization of MSCs (3), the wide availability of tissues and therefore derived cells, allows the development of a pool of MSCs of allogeneic origin for cryopreservation with abundant reserves for their use and commercialization. Application of an intramammary treatment method for allogeneic fetal MSCs for mastitis (4 and 5). Consequently, there is a decrease in the use of antibiotics in order to treat mastitis (6).
[0052] To determine the in vivo effects of MSCs—which should be dependent on the population of stem cell origin used, BM-MSCs and AT-MSCs were compared in terms of proliferation, immunomodulation and lymphocyte activation potentials. The in vivo effects of MSCs were also dependent on the tissue into which they were inoculated. The potential for migration (“homing”) and implantation (“engraftment”) to the target tissue was evaluated by identifying fluorescently labeled MSCs in the parenchyma of the mammary gland.
[0053] The present invention relates to a suspension of mesenchymal stem cells (MSC) of fetal and allogeneic origin for the treatment of mastitis in dairy animals, including bovines. The ability to migrate to the site of injury allows the treatment of mastitis, strengthening the immune response, exerting antimicrobial action and subsequently promoting the regeneration of damaged glandular tissue. The present composition also relates to a composition comprising such a suspension of allogeneic bovine MSCs derived from fetal tissues. This product can be stored by cryospreservation and can be administered intramammary.
[0054] The present invention would be a substitute or complementary alternative for the use of antibiotics, increasing the effectiveness of the treatment and reducing the risk that their excessive use represents for public health. Currently, there is no such tool available for the treatment of mastitis in dairy animals, including cattle, thus generating great therapeutic, productive and animal welfare benefits with the present invention.
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[0056] Trials were conducted to evaluate efficacy parameters of intramammary administration of the allogeneic composition of fetal bovine MSC in cows with induced mastitis.
[0057] In the examples below—unless otherwise stated, trials were conducted using 15 clinically healthy (free from Brucellosis, Tuberculosis and Leukosis) Holstein Friesian cows of approximately 2 years of age. Three bacterial milk cultures were performed to rule out the presence of intramammary infections. Cows were milked twice daily using a separate milking unit and milk production was quantified after each milking. The trial was conducted according to the Applicant's Bioethics guidelines. The animals were kept under the same management and were fed alfalfa and concentrate.
Example 1: Preparation and Administration of the Inoculum
[0058] A strain of S. aureus was thawed and seeded on blood agar and incubated at 37° C. in aerobiosis overnight. A bacterial colony of S. aureus was then incubated in 5 mL of brain heart broth in a 15 mL tube for 10 hours at 37° C. Subsequently, eight dilutions of the broth were prepared in PBS and 100 uL of each dilution were seeded in duplicate blood agar. Using a sterile rake, the inoculum of each dilution was distributed on the surface of each plate. The plates were incubated overnight in aerobiosis at 37° C. Subsequently, the colonies on the plate containing between 30-300 CFU were counted in duplicate and averages were obtained. To calculate the number of CFU per mL, the equation (Eq. 1) was used:
Number of colonies×(10).sup.A(number of the dilution+1)=Amount of CFU/mL (Eq. 1)
[0059] The volume necessary to obtain a bacterial suspension with a concentration of 1000 CFU/mL in 1 Lt of PBS was calculated using the equation (Eq. 2):
C1×V1=C2×V2 (Eq. 2)
[0060] where C1=Concentration expressed in CFU/mL, V1=Volume to be extracted from the incubated broth, C2=Desired concentration (1000 CFU/mL) and V2=Total volume of the suspension for inoculate (1000 mL).
Example 2: Challenge
[0061] The cows (n=15) at approximately 30 days postpartum were challenged by inoculation of the two left mammary quarters via the intracisternal route administration of 1 mL of a 1000 cfu/mL suspension of S. aureus. 1 mL of PBS was inoculated into the right quarters as a control.
[0062] From the day of the challenge and daily for 15 days, the cows were clinically evaluated and milk samples were collected to determine SCC and CFU. The clinical evaluation and the collection of milk samples were carried out as detailed below.
[0063] Experimental Design
[0064] The cows were randomly assigned to three experimental groups of 5 animals each. All the animals were challenged by inoculation of the two left mammary quarters via the intracisternal route using 1 mL of a 1000 CFU/mL suspension of S. aureus.
[0065] 1 mL of PBS was inoculated into the right quarters as a control. The NEG group (without therapy) was treated on days 4 and 10 with a 5 mL Ringer Lactate solution via the teat canal in the two infected quarters. The ATB group (antibiotic therapy) was treated with a commercial suspension of 50 mg of pirlimycin on experimental days 4 and 5 via the teat canal in the two infected quarters. The MSC group (MSC therapy) was administered with a 2.5×10.sup.7 suspension of MSC in 3 ml of Lactated Ringer via the teat canal in the two infected quarters. From the day of the challenge and daily during 15 days, the cows were clinically evaluated and milk samples were collected daily to determine SCC and CFU (
[0066] Clinical Examination
[0067] From the day of the challenge and daily during 15 days, the cows were evaluated by clinical examination that included determination of heart rate, respiratory rate, ruminal rate, rectal temperature, and mucous coloration. According to the classification table provided below (Table 1.; Wenz J, Garry F, Barrington G. 2006. Comparison of disease severity scoring systems for dairy cattle with acute coliform mastitis. JAVMA. 229, 259-262). From two days prior to the challenge and daily, the udder of each cow was evaluated after each milking by manual palpation of each mammary quarter to detect abnormalities such as volume increase, pain, redness, and temperature increase (Table 1). The abnormalities in the appearance of the milk were evaluated at the beginning of milking using a strip cup with dark surface to classify the secretion as normal, watery, viscous, with the presence of lumps, blood or pus (
TABLE-US-00001 TABLE 1 Dairy cow clinical signs severity classification system based on systemic signs of the disease. Variable Criteria Score Rectal temperature (° C.) 37.8-39.27 0 39.33-39.8 1 >39.8 o <37.8 2 Hydratation status (degree None 0 of enophthalmos) Mild 1 Moderate 2 Marked 3 Rumen contraction rate ≥2 0 (contractions/min) 1 1 0 2 Attitude (signs of None 0 depression) Mild 1 Marked 2 Cows with total score of 0 to 2 were classified as having mild disease, cows with total score of 3 to 5 were classified as having moderate disease, and cows with total score of 6 to 9 were classified as having severe disease.
TABLE-US-00002 TABLE 2 Classification system for the severity of clinical signs in the udder of dairy cows based on local signs of the disease. Variable Criteria Score Quarter firmness None 0 Firm 1 Very Firm 2 Quarter swelling None 0 1.5 times 1 2 times 2 Signs of quarter pain None 0 Present 1 Secretion characteristics Normal 0 Thin with clots and flakes 1 Serum-like with clots or flakes 2 Cows with total score of 0 to 2 were classified as having mild disease, cows with total score of 3 or 4 were classified as having moderate disease, and cows with total score of 5 to 7 were classified as having severe disease.
[0068] Bacteriological Analysis (CFU)
[0069] Milk samples prior to milking were collected aseptically from all cows following the protocols of the International Dairy Federation (1985) on three consecutive days prior to the experimental challenge (
[0070] Somatic Cell Count (SCC)
[0071] From two days prior to the challenge and daily for the 15 days of the study, milk samples were collected prior to milking from all cows in 50 ml plastic tubes with bronopol as preservative. SCC was performed on the milk samples using a Fossomatic flow cytometer (
[0072] Obtaining and Cultivating MSC
[0073] MSCs were isolated from bovine fetal adipose tissue. Bovine fetuses at the last third of gestation were obtained from an abattoir and transported in a thermal container to the laboratory. Fetal adipose tissue was extracted by incision in the abdominal midline and surgical extraction using a scalpel and sterile forceps. Then the adipose tissue was washed with phosphate buffer solution (PBS), pH 7.4 and sectioned using a scalpel. Subsequently, the tissue was incubated at 38° C. for 45 min under constant agitation in a digestion medium consisting of 0.5% collagenase diluted in HBSS solution (Hank's Balanced Salt Solution). The enzymatic reaction was stopped by adding expansion medium consisting of DMEM medium (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, 500 uL of amphotericin B and 500 uL of penicillin/streptomycin in an amount equivalent to the digestion medium. Subsequently, the tissue was filtered using pores of 40 um and centrifuged twice at 400×g for 5 min using washing medium, which consisted of DMEM supplemented with 100 ug/mL of amphoterecin B, 100 IU/mL of penicillin and 100 ug/mL streptomycin. The pellet was resuspended in expansion medium, and then transferred to 175 cm.sup.2 cell culture flasks. MSC cultures derived from adipose tissue were incubated at 38° C. under a humid atmosphere with 5% CO.sub.2. After 48 hours, the cells not adherent to the plastic were removed by changing the culture medium. Upon reaching 80 to 90% confluence, MSC passages were performed by removal with trypsin/EDTA, supplemented with 250 u.Math.g/mL of amphoterecin B, 100 IU/mL of penicillin and 100 u.Math.g/mL of streptomycin.
[0074] Statistical Analysis
[0075] The data on clinical variables, SSC and CFU were analysed using the Shapiro Wilks normality test. Treatment effects were evaluated by Kruskal Wallis analysis. The experimental days and the treatment were evaluated as independent variables, and the clinical variables, the SCR and the CFU were evaluated as dependent variables. A significance value (P<0.05) was used. All statistical analyzes were performed using the Info Stat software (Cordoba, Argentina, 2008).
[0076] Comparative Analysis of Clinical Variables in Cows Challenged with S. aureus
[0077] The cows showed an increase in volume of the challenged quarters from day 2 post-challenge. No traces of blood or pus were observed in the milk by strip cup with dark surface testing but lumps were detected in samples from all cows intermittently during the study. The average values of the clinical variables evaluated including heart rate, respiratory rate, ruminal rate, rectal temperature, thermography of the mammary quarters, and milk production did not show differences (P>0.05) between the experimental groups (Table 3). The average daily milk production of the NEG, ATB and MSC groups was 17.3±1.8; 16.7±0.8 and 14.7±1 I, respectively. There was a decrease in milk production between days 5 and 7 of the study of 6.8; 2.1 and 2.3 (NEG, ATB and MSC Groups, respectively).
TABLE-US-00003 TABLE 3 Clinical and productive variables in cows (n = 5 each group) challenged with S. aureaus and assigned to experimental groups NEG (no therapy), ATB (antibiotic therapy) and MSC (therapy with MSC). Variable/Treatment group NEG ATB MSC Heart rate (bpm) 59.2 ± 3.8 53.3 ± 3,5 53.1 ± 1.6 Respiratory rate (bpm) 30.9 ± 3.4 28.6 ± 2.5 30.5 ± 1.9 Ruminal frequency (in 2 min) 2.9 ± 0.3 2.6 ± 0.3 2.4 ± 0.2 Rectal temperature (° C.) .sup. 38 ± 0.7 38.1 ± 0.3 37.9 ± 0.2 Termography FL (° C.) 34.1 ± 0.8 33.8 ± 0.7 34.4 ± 0.7 Termography BL (° C.) 34.2 ± 2.5 34.3 ± 0.6 .sup. 34 ± 0.6 Termography FR(° C.) 33.8 ± 0.9 33.7 ± 0.7 33.6 ± 0.5 Termography BR (° C.) 34.1 ± 1.2 34.2 ± 0.5 .sup. 34 ± 0.4 Milk yield (Its.) 17.3 ± 1.8 16.7 ± 0.8 14.7 ± 1.sup. Milk yield reduction days 5-7 (Its.) 6.8 2.1 2.3 Values represents means ± standard deviations
[0078] Comparative Analysis of RCS in Cows Challenged with S. aureus
[0079] From the day of the challenge and daily for 15 days, milk samples were collected to determine SCC. The SCC values were subsequently expressed as a linear score (LS-SCC) by means of logarithmic transformation (LS=LOGN). The mammary quarters of the NEG and ATB groups (without therapy and antibiotic therapy, respectively) obtained statistically similar LS-SCC values compared to the uninfected quarters from day 1 to day 10 post-challenge (
[0080] Comparative Analysis of CFU in Cows Challenged with S. aureus
[0081] From the day of the challenge and daily for 15 days, milk samples were collected for determination of CFU. The CFU values were subsequently expressed as a linear score (LS-CFU) by means of logarithmic transformation (LS=LOGN). Milk samples from the MSC group reached higher (P<0.05) PL-CFU (4.6) values compared with the samples obtained from the ATB group quarters (−0.7) on day 3 post-challenge (
[0082] The previous example demonstrates that the cows challenged with S. aureus and assigned to the MSC group reached the highest values of SCC and CFU (Days 2 and 3, respectively), after the challenge with S. aureus but prior to the administration of MSC. However, the group of cows treated with MSC significantly decreased SCC values on day 10 post-challenge. Furthermore, the group of cows treated with MSC reached intermediate values of CFU without showing significant differences compared to the groups without therapy and with antibiotic therapy. These data confirm that MSCs exerted an immunomodulatory and antibacterial effect in cows challenged with S. aureus.
Example 3 Antibacterial Potential of MSCs Derived from Bone Marrow and Fetal Bovine Adipose Tissue
[0083] Strains ATCC 25923 and SAU-1 S of S. aureus were used in order to determine their survival in unconcentrated MSC conditioned medium (CM), concentrated MSC conditioned medium (CCM), MSC conditioned medium activated by pre-exposure of S. aureus (ACM). In these same MSC cultures, the mRNA levels of the antimicrobial peptides b-defensin 4A (bBD4A) (
[0084] The CM were obtained from BM-MSCs, AT-MSCs and FBs cultures as control. Cells were seeded at a concentration of 6×10.sup.3 cells/cm.sup.2 until reaching 70 to 80% of confluency. Subsequently, MSCs and FBs were cultured in DMEM medium with 4500 mg/L D-Glucose (Mediatech, Incorporated) without serum, antibiotic and antifungal for 72 h at 38° C. under a humid atmosphere with 5% CO.sub.2. Next, the CM were collected and then centrifuged at 400×g for 5 min and later filtered through pores of 0.22 μm and frozen at −20° C. until use. For the experiments with concentrated CM, the medium was concentrated 10 times its initial volume, by ultrafiltration using Amicon Ultra filters (Merck Millipore, Corporation, Cork, Ireland) with a 3 kDa NMWL membrane according to the manufacturer's instructions. The concentrated media were then frozen at −20° C. until used. Subsequently, the antibacterial potential of the activated CM obtained from cultures of BM-MSC, AT-MSCs, as well as FBs that were previously exposed to 300 CFU of the local strain SAU-1 S of S. aureus by a 6 h period (
[0085] Consequently, the above tests indicate that BM-MSCs and AT-MSC CM possess antibacterial effect against S. aureus in an in vitro culture system. This antibacterial potential is increased by concentrating the media by filtration and activating the CM by pre-exposure of MSCs to S. aureus. The results suggest that the antibacterial effect of MSCs is mediated by the expression of antimicrobial peptides NK1 and bBD4A. See
Example 4 Determination of the Proliferation Potential of Fetal MSCs Derived from Bone Marrow and Bovine Adipose Tissue
[0086] Isolation of MSC from fetal bone marrow (BM-MSCs). Bone marrow was obtained from femurs of bovine fetuses of 8 to 9 months of gestation (n=5) derived from slaughterhouse (
[0087] The suspension obtained was centrifuged at 400 G for 10 minutes at RT. Subsequently, the BM-MSC-MOs and AT-MSCs were seeded separately in expansion medium consisting of DMEM supplemented with 5% fetal bovine serum (FBS). Upon reaching 80 to 90% cell confluence, passages were made by stirring with 0.25% Trypsin/EDTA for 5 min. The MSCs were cultivated in expansion medium until reaching 80% confluence.
[0088] The proliferation potentials of bovine BM-MSCs and AT-MSC were compared by calculating population doubling (DP) according to tissue of origin. Population doublings were determined. BM-MSCs and AT-MSCs were seeded separately in triplicate in 3.5 cm culture plates in expansion medium until reaching 70 to 80% confluence. Then the MSCs were passed at a concentration of 8.4×10.sup.3 cells/cm.sup.2. Cells from passages 1 to 15 were maintained with a fixed subculture period of 5 days and counted and reseeded. The cell count data were used to estimate the population doubling and to construct an accumulated population doubling by means of the equation (Eq. 1):
DP=ln(Nf/Ns) (Eq. 1)
[0089] Where ln is the natural logarithm, Nf is the number of cells obtained in the subculture and Ns is the number of cells initially seeded (Freshney R I. 2005. Culture of Animal Cells: A Manual of Basic Technique. 5th edition. Editorial Wiley-Liss. New Jersey. 38-358). To establish a proliferation curve with the total cumulative number of cells during the passages, the equation (Eq. 2) was used:
Number of total cells of P.sub.n=Number counted in P.sub.n×(Number counted at Pn-i/Total number of cells seeded) (Eq. 2)
[0090] The proliferation potential was determined at two concentrations (5000 and 8500 cells/cm.sup.2) for 15 days (
[0091] The proliferation and immunogenicity potentials of MSCs are dependent on the source of the tissue of origin, since MSCs derived from bone marrow possess high proliferation and immunogenicity potentials compared to MSCs derived from adipose tissue. BM-MSCs have greater proliferation potential, due to the fact that they double their population in less time compared to AT-MSCs from bovine fetuses. However, both BM-MSCs and AT-MSCs possess similar immunomodulation potentials.
Example 5 Determination of the Immunomodulatory Potential of BM-MSCs and AT-MSCs by Gene Expression Analysis of Paracrine Immunomodulatory Factors (IDO, IL-6, IL-10, PGE.SUB.2 .and TGFβ1) and Determination of the Enzymatic Activity of IDO in Conditioned Medium of MSC Preactivated with IFNγ
[0092] mRNA levels of paracrine immunomodulatory factors in BM-MSCs and AT-MSCs preactivated with IFNγ. The immunomodulatory potential was evaluated by quantifying the expression of anti-inflammatory genes (IDO, IL-4, IL-10) in BM-MSCs and AT-MSCs treated with 10, 20 or 40 ng/mL of IFNγ. (
[0093] The Q-PCR data obtained were analyzed using the comparative method of Ct, in relation to the Ct of the endogenous genes GAPDH and p-ACTIN (Schmittgen T D, Livak K J. 2008. Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc 3, 1 101-1 108). Determination of the enzymatic activity of IDO in conditioned medium of MSCs preactivated with IFNγ. BM-MSCs and AT-MSCs from 2nd to 3rd passage were seeded at a concentration of 1×10.sup.4 cells/cm.sup.2 in high glucose DMEM medium supplemented with 10, 20 and 40 ng/mL of IFNγ (R&D Systems, Minneapolis, USA) for 72 h. Subsequently, the conditioned medium of MSC was collected to determine the enzymatic activity of IDO. For this purpose, the N-formyl-kinurenine concentration in the medium was quantified using a colorimetric assay (Saulnier N, Loriau J, Febre M, Robert C, Rakic R, Bonte T, Buff S, Maddens S. 2016. Canine placenta: A promising potential source of highly proliferative and immunomodulatory mesenchymal stromal cells? Vet Immunol Immunopathol 171, 47-55) and comparing it with a D-Quinurenine concentration curve (Sigma-Aldrich, USA). Once collected, the conditioned medium was centrifuged at 750 g for 5 min and filtered through pores of 0.22 μm. A 200 μl aliquot of MSC conditioned medium was mixed with 100 ml of 30% trichloroacetic acid (Sigma-Aldrich, USA) and the mixture was incubated at 50° C. for 30 minutes. Once centrifuged at 15,000 g for 1 min, 100 μl of supernatant was transferred to each well of a 96-well flat-bottom plate and 100 μl of Ehrlich reagent (100 mg of p-dimethylamino benzaldehyde diluted in 5 ml of acid glacial acetic). Next, the optical density at 450 nm was determined with a microplate reader (Model 3550, Bio-Rad, Arizona, USA). See
[0094] BM-MSCs and AT-MSCs treated with 20 and 40 ng/mL of IFNγ expressed (4.1, 1, 6 and 2.5, 1.4 times, respectively) higher (P<0.05) levels of mRNA of IL-6 in relation to the control MSC treated with 10 ng/mL of IFNγ (
[0095] Additionally, the expression levels of major histocompatibility complex immunogenicity genes I and II (MHC-I and II) and T lymphocyte costimulatory molecules (CD80 and CD86) were quantified (
[0096] These results indicate that both MSC cell types express immune mediating factors that could exert an immunomodulatory role for therapeutic treatment in cattle. Considering these results, it is possible to conclude that the immunogenicity potential of MSCs is dependent on the source of tissue origin, since BM-MSCs present a higher expression of MHC-I I compared to AT-MSCs, which could determine a higher Immune rejection in a potential application in allogeneic therapy.
Example 6 Determination of the Lymphocytic Activation Potential of Fetal Bovine MSCs by Mixed Culture
[0097] The effect of conditioned medium of BM-MSCs and AT-MSCs on the proliferation of T lymphocytes activated by alloantigens in vitro was evaluated by means of mixed leukocyte reaction (MLR) (Bocharov G, Luzyanina T, Cupovic J, Ludewig B. 2013. Asymmetry of cell division in CFSE-based lymphocyte proliferation analysis. Frontiers in immunology 264, 1-7). Bovine peripheral blood mononuclear cells (PBMC) were isolated by density gradient (Flistopaque-1077, Sigma-Aldrich). A 15-mL sample of bovine blood was drawn through the coccygeal vein using heparin vacutainer tubes. Then the blood was mixed with PBS in equal parts (1:1). On the other hand, in a 50 mL falcon tube, 15 mL of Histopaque (Sigma-Aldrich) were added and the blood solution with PBS (30 mL) was added without mixing. Subsequently, the tube was centrifuged at 900 g without brake for 35 minutes. In this way, a separation into four phases was achieved, plasma plus PBS, PBMCs, Histopaque and in the background granulocytes and red blood cells. With a pipet, the PBMC phase was extracted, then it was washed with 10 mL of PBS and centrifuged twice at 1000 g for 10 minutes, to then be re-suspended in supplemented RPMI. In order to perform the purification of peripheral blood lymphocytes (PBLs), the PBMCs were re-suspended in RPMI 1640 medium with 10% FBS (Hyclone Laboratories), 100 μg/mL of amphotericin B, 100 μg/mL of streptomycin and 100 IU/mL of penicillin in a 6-well plate (Costar 3516, USA) and incubated at 38° C., with 5% CO.sub.2 for 2 h. After that, the cells not adherent to the plastic were removed and washed with PBS. The PBL was centrifuged and the pellet was resuspended in PBS (Quah et al., 2007). Previously, cell counting was performed in a Neubauer chamber using trypan blue to determine cell viability. Subsequently, the cell concentration was adjusted to 2×10.sup.6 cells per mL. The PBLs was subsequently labelled with a fluorescent carboxyfluorescein succinimidyl ester (CFSE) probe using a cell proliferation kit (CellTrace, Thermo Fisher, California, USA). Each vial of stock solution (5 mM) was diluted with 18 pL of DMSO and from this dilution 2 μL (10 pM) were used to mark a total of 1×10.sup.6 PBLs in 1 mL of PBS in a suspension that was incubated for 20 min at 37° C. by gentle shaking, protected from light (Quah et al., 2007; Bocharov G, Luzyanina T, Cupovic J, Ludewig B. 2013. Asymmetry of cell division in CFSE-based lymphocyte proliferation analysis. Frontiers in immunology 264, 1-7). Subsequently to this solution a five-fold proportion of medium with SFB was added and it was incubated again for 5 minutes, this process is carried out to eliminate the free dye that is in the solution. It was then centrifuged and the pellet was resuspended in culture medium for subsequent tests. The PBMCs was treated with mitomycin C to inhibit DNA synthesis, suppressing its proliferation but keeping the cells viable and capable of stimulating the proliferation of PBLs. For this, 1×10.sup.6 PBMCs were previously treated with 50 μg/mL of mitomycin C at 37° C. for 2 h and then they were washed three times with PBS with 5% FBS. To induce alloproliferation, PBLs labeled with CFSE (CellTrace) as responder were co-incubated with PBMCs as allogeneic stimulator treated with mitomycin C in a 96-well U-bottom culture plate for 5 days (Bocharov G, Luzyanina T, Cupovic J, Ludewig B. 2013. Asymmetry of cell division in CFSE-based lymphocyte proliferation analysis. Frontiers in immunology 264, 1-7). Different stimulator and responder ratios (1:1, 1:2 and 1:4) were incubated with the aim of standardizing the method for cattle and working with the proportion that stimulates the proliferation of PBLs to the highest level. As a proliferation control, PBMCs plus PBLs without conditioned medium was used. The cells were subsequently recovered for proliferation analysis by flow cytometry. The data were analyzed using a Gallios cytometer kit (Beckman Coulter, Brea, Calif., USA) and the Cell Quest program. See
Example 7 Migration Potential and Stimulation of Angiogenesis In Vitro of MSCs Derived from Bone Marrow and Bovine Fetal Adipose Tissue
[0098] The “scratch” migration test was used to determine the migratory capacity of MSCs, using fetal FBs as biological controls (
[0099] Transwell migration analysis was used to determine the potential for cell migration in response to chemotactic stromal cell-derived factor-1 (SDF-1) (
[0100] The potential between BM-MSCs and AT-MSCs was similar, which can be associated to similar expression levels of the CXCR4 receptor. The angiogenic potential of the MSC and FB conditioned medium was evaluated using the in vitro endothelial cell tubule formation assay (
[0101] Once they reached 80% confluence, the culture medium was removed and DMEM without SFB was added in order to produce conditioned medium. Once collected, the conditioned medium was concentrated 10 times by centrifugation for 30 minutes at 1800 rpm in Amicon Ultra Centrifugal filters (Merck Millipore, Tullagreen) and was subsequently frozen at −20° C. until use. For the tubule formation assay, 100 ml of matrigel without growth factors (Corning, Bedford) was pipetted into 24-well plates for the establishment of a basement membrane matrix. The matrigel was gelled at 38° C. for 30 minutes. Subsequently, the endothelial cells (1.8×10.sup.5 cells/well) previously extracted were seeded and cultured on top of the gelled Matrigel membrane. These cells were then resuspended in the concentrated medium of BM-MSCs, AT-MSCs and FBs and seeded on the Matrigel layer. After 6 hours of incubation at 38° C., the tubules formed were quantified by obtaining digital images obtained with 4× magnification and evaluation using Image J software (NIMH). The concentrated AT-MSC conditioned medium induced greater (P<0.05) tubule formation, compared to BM-MSCs, FBs and their DMEM controls with 5% SFB and DMEM (
Example 8: Evaluation of Safety Parameters Including Clinical Variables, Lymphocyte Activation and Cytokine Production in Trials of Intramammary Administration of an Allogeneic Therapy of Fetal Bovine MSCs in Healthy Holstein Friesian Heifers
[0102] A safety test was carried out in healthy cows in order to determine the effect of inoculation, via the teat canal, of an MSC suspension of allogeneic origin on the clinical parameters including hemogram and biochemical profile in Holstein Friesian cows. MSCs were isolated from adipose tissue collected from abattoir-derived fetuses previously tested for Brucella abortus (Rose Bengal), Leptospirosis (Microagglutination), Bovine Viral Diarrhea (Elisa) and Infectious Bovine Rhinotracheitis (Elisa). Only fetuses negative for these diseases were used as donors (n=14). The safety test was carried out in a dairy farm of 997 lactating cows that has been declared free of Brucellosis, Tuberculosis and Bovine Leukosis. Clinically healthy Holstein Friesian heifers (n=8) of age (15.4±1.9 months) with weight (445.6±37.3 kg) were used. A total of 25×10.sup.6 MSCs were suspended in 3,000 Ringer Lactate and this suspension was inoculated into each of two mammary quarters of each cow. A control suspension of 3 mL of Lactated Ringer was inoculated in each of the two remaining mammary quarters of each cow. The order of administration per room was determined randomly. Two doses of MSCs were administered 10 days apart (dose 1=day 0 and dose 2=day 10) and clinical parameters (heart rate, respiratory rate, ruminal rate, rectal temperature and mammary gland temperature) were evaluated daily from the day 0 to day 20 (Table 4).
[0103] In addition, blood samples were obtained from the coccygeal vein every 5 days from day 0 to day 20, for hemogram analysis and biochemical profile. Additionally, lymphocyte and monocyte populations were isolated from peripheral blood for analysis of CD3, CD4, CD8, and FoxP3 markers by quantitative PCR.
TABLE-US-00004 TABLE 4 Clinical, hematological, and biochemical profile parameters in Holstein Friesian heifers inoculated with two doses of adipose-derived fetal bovine MSC intramammary. Variable Expected value Obtained value HR (bpm) 60 adult 120 calf 77.7 ± 4.5 RR (bpm) 24 adult 48 calf 45.5 ± 9.4 Ruminal frequency (in 2 3 3.2 ± 0.5 min) Rectal Temp. 38-39 adult y 39-40.5 ternero 38.9 ± 0.3 Termography FL (° C.) 33.45-34.1 31.8 ± 2.2 Termography BL (° C.) 33.45-34.1 32.2 ± 2.1 Termography FR (° C.) 33.45-34.1 32.1 ± 1.8 Termography BR (° C.) 33.45-34.1 32.3 ± 1.7 Erythrocytes (mm3) 5,000,000-10,000,000/mm.sup.3 6,213,214 ± 138,603 VGA (%) 24-46% 28.9 ± 0.7 Hemoglobin (g/dL) 8-15 g/dL .sup. 10 ± 0.3 Leukocytes (mm.sup.3) 4,000-12,000/mm.sup.3 23,491 ± 9,647 Eosinophils (mm.sup.3) <2,400 mm.sup.3 267.2 ± 113.7 Basophils (mm.sup.3) <200 mm.sup.3 0 Myelocytes (mm.sup.3) <0 mm.sup.3 0 Juveniles (mm.sup.3) <0 mm.sup.3 0 Baciliformes (mm.sup.3) <120 mm.sup.3 0 Segmented (mm.sup.3) 600-4,000 mm.sup.3 7,919 ± 538 Lymphocytes (mm.sup.3) 2,500-7,500 mm.sup.3 11,328 ± 2,758 Monocytes (mm.sup.3) 25-850 mm.sup.3 0 No Platelets(mm.sup.3) 100,000-800,000 mm.sup.3 259,507 ± 45,106 TPP (g/dL) 6.5-7.5 g/dL 6.7 ± 0.03 Albumins (g/dL) 3.0-3.6 g/dL 3.21 ± 0.04 Globulins (g/dL) 3.0-3.5 g/dL 3.48 ± 0.06 A/G Index 0.86-1.2 0.93 ± 0.03 Cholesterol (mg/dL) 80-120 mg/dL 106.29 ± 8.5 Calcio (mg/dL) 9.7-12.4 mg/dL 8.64 ± 0.1 Phosphorus (mg/dL) 5.6-6.5 mg/dL 6.57 ± 0.3 Glucose (mg/dL) 45-75 mg/dL 68.08 ± 18.1 BUN (mg/dL) 20-30 mg/dL 14.1 ± 5.9 Creatinine (mg/dL 1-2 mg/dL 0.81 ± 0.04 CK total (U/L) 14-107 U/L 265.67 ± 38 AST (U/L) 78-132 U/L 71.78 ± 4.2 GGT (U/L) 6.1-17.4 21.5 ± 0.8 AP (U/L) <488 U/L 352.72 ± 25.9 Total Bilirubin (mg/dL) 0.01-0.5 mg/dL 0.16 ± 0.04 Fibrinogen (mg/dl) 300-700 mg/dL 312.74 ± 16.8
[0104] The analysis of the data indicates that the administration of a 25×10.sup.6 suspension of allogeneic MSCs via the teat canal, in two doses separated by ten days, does not significantly affect the clinical parameters, hemogram variables and biochemical profile and expression of markers. of CD4+ and CD8+ lymphocyte populations or on the mRNAs of the CD25 and CD62L lymphocyte activation molecules (