Methods for assessing the purity of a mesenchymal stem cells preparation

Abstract

The present invention relates to a method for assessing, evaluating and/monitoring the purity of a mesenchymal stem cells preparation, in particular of an adipose stem cells preparation, comprising measuring the expression level of at least one growth factor.

Claims

1. An in vitro method for preparing a substantially pure cell preparation of mesenchymal stem cells (MSCs) suitable for use as a cell therapy product in regenerative medicine, wherein said method comprises the steps of (i) providing a cell culture medium of a cell preparation comprising MSCs isolated from tissues, (ii) measuring the expression level of at least one growth factor secreted by said cell preparation into the cell culture medium, wherein said at least one growth factor is SDF-1 α and/or VEGF, and (iii) selecting the cell preparation as being substantially free of contaminating fibroblasts when the SDF-1α and/or VEGF expression levels are at least 80% of a reference expression level of SDF-1α and/or VEGF of a pure MSC preparation isolated from the same tissue and cultured in the same conditions as said cell preparation, wherein the SDF-1α expression level is at most 100 pg/ml and/or the VEGF expression level is at least 90 pg/ml, and wherein the expression levels are measured at the protein level, thereby preparing a substantially pure MSC preparation suitable for use as a cell therapy product in regenerative medicine.

2. The in vitro method according to claim 1, wherein said mesenchymal stem cells are isolated from tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver.

3. The in vitro method according to claim 1, wherein said mesenchymal stem cells are adipose stem cells (ASCs).

4. The in vitro method according to claim 1, wherein said cell preparation is selected as being substantially pure when the SDF-1α expression level is of at most 100 pg/ml and/or the VEGF expression level is of at least 200 pg/ml in the cell culture medium, and wherein said cell preparation is cultured in hypoxic conditions and at high concentration of glucose, before measuring the expression level.

5. The in vitro method according to claim 1, wherein said cell preparation is selected as being substantially pure when the SDF-1α expression level is of at most 100 pg/ml and/or the VEGF expression level is of at least 90 pg/ml in the cell culture medium, and wherein said cell preparation is cultured at tissular oxygen tension and at high concentration of glucose, before measuring the expression level.

6. The in vitro method according to claim 1, wherein said expression level is measured at the protein level and wherein said measure is the detection and/or quantification of said at least one growth factor secreted in the cell culture supernatant.

7. The in vitro method according to claim 4, wherein said cell preparation is cultured at about 0.1% O2 and at about 4.5 g/l of glucose, before measuring the expression level.

8. The in vitro method according to claim 5, wherein said cell preparation is cultured at about 5% O2 and at about 4.5 g/l of glucose, before measuring the expression level.

9. The in vitro method according to claim 4, wherein said cell preparation is selected as being substantially pure when the SDF-1α expression level is of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and/or the VEGF expression level is at least 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289 or 290 pg/ml in the cell culture medium.

10. The in vitro method according to claim 5, wherein said cell preparation is selected as being substantially pure when the SDF-1α expression level is of at most 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 pg/ml; and/or the VEGF expression level is of at least 95, 100, 105, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 pg/ml in the cell culture medium.

11. The in vitro method according to claim 1, wherein said cell preparation of MSCs isolated from tissues are cultured in vitro for one or more passages prior to providing the cell culture medium for measuring the expression level of at least one growth factor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph showing ASC and DF in proliferation medium (A) and in osteogenic differentiation medium (B).

(2) FIG. 2 is a graph showing the cell proliferation of ASC and DF according to the number of passages.

(3) FIG. 3 is a histogram showing the cell survival of ASC and DF in proliferation medium without FBS, at 0.1 or 5% O.sub.2.

(4) FIG. 4 is a set of histograms showing KGF secretion (A), b-FGF secretion (B), IGF-1 secretion (C), and HGF secretion (D) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at 0.1 or 5% O.sub.2.

(5) FIG. 5 is a set of histograms showing VEGF secretion (A) and SDF-1α secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at 0.1% O.sub.2.

(6) FIG. 6 is a set of histograms showing VEGF secretion (A) and SDF-1α secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at 5% O.sub.2.

(7) FIG. 7 is a histogram showing SDF-1α secretion of 5 different ASC/DF dilutions in proliferation medium with 4.5 g/l glucose, at 21% O.sub.2.

(8) FIG. 8 is a set of histograms showing VEGF secretion (A) and SDF-1α secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 1 g/l glucose, at 0.1% O.sub.2.

(9) FIG. 9 is a set of histograms showing VEGF secretion (A) and SDF-1α secretion (B) of 5 different ASC/DF dilutions in proliferation medium with 1 g/l glucose, at 5% O.sub.2.

(10) FIG. 10 is a histogram showing SDF-1α secretion of 5 different ASC/DF dilutions in proliferation medium with 1 g/l glucose, at 21% O.sub.2.

EXAMPLES

(11) The present invention is further illustrated by the following examples.

Example 1

(12) Materials and Methods

(13) This study was performed according to the guidelines of the Belgian Ministry of Health. All procedures were approved by the Ethical Committee of the Medical Faculty (Université Catholique de Louvain) for tissue procurement and clinical study (B40320108280). All materials were obtained from Lonza (Verviers, Switzerland), Sigma-Aldrich (St. Louis, Mo., USA), or Invitrogen (Carlsbad, Calif., USA) unless otherwise noted.

(14) ASC and DF Isolation and Culture

(15) A combined harvesting of human adipose (mean: 7.4 g) and dermal (mean: 1.5 cm.sup.2) tissues were performed in 8 patients (Table 1) undergoing elective plastic surgery after informed consent and serologic screening, by lipoaspiration using the Coleman technique, and skin biopsy, respectively. Adipose tissue and skin samples were kept in sterile conditions for a maximum of 60 minutes at 4° C. before adipose-derived stem cells (ASC) and dermal fibroblasts (DF) isolation.

(16) TABLE-US-00001 TABLE 1 Coupled ASC/DF donors characteristics Donor 1 2 3 4 5 6 7 8 Age (years) 19 44 40 62 56 46 45 41 Sex F F F F F F F F Clinical mammaplasty abdominoplasty mammaplasty Lat. abdominoplasty mammaplasty mammaplasty mammaplasty indication Dorsi flap

(17) The adipose tissue was digested with collagenase (1/2 w/v) in a water bath at 37° C. for 60 minutes. Collagenase was inactivated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. Collected tissue was centrifuged for 10 minutes at 1500 rpm at room temperature. The pellet was suspended in a proliferation medium made up of DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μl/ml amphotericin B) and filtered through a 500-μm mesh screen. The collected suspension was then seeded in 25 cm.sup.2 culture flasks with proliferation medium.

(18) DF were isolated by extraction from de-epidermized dermal biopsies, minced in 2 mm×2 mm fragments and placed in plastic well. Small volume of the proliferation medium was added to avoid detachment from the plastic surface.

(19) After 24 hours of incubation at 37° C. and 5% CO.sub.2, the proliferation media were replaced. This initial passage of the primary cells is referred to as passage 0. Dermal pieces were removed from the culture dish when adherent cells were visible on the plastic surface surrounding tissue fragments. Cells were maintained in proliferation medium (changed 2 times/week) up to passage 4, after sequential trypsinizations. Cells from 3 donors were cultivated until passage 15 to study the proliferation profile in standard culture conditions (37° C., 21% O.sub.2, 5% CO.sub.2, 4.5 g/l glucose).

(20) Membrane Marker Profile Characterization

(21) At passage 4, ASC and DF were characterized for standard cell surface markers (CD44, CD45, CD73, CD90, CD105, stro-1, CD106, CD146, CD166, CD19, CD31, CD11b, CD79α, CD13, HLA-DR, CD14, CD34) [Dominici et al., Cytotherapy. 2006; 8(4):315-317; Bourin et al., Cytotherapy. 2013; 15:641-648] by fluorescence-activated cell sorting (FACScan; BD Biosciences, San Jose, Calif.).

(22) Briefly, ASC were stained with saturating amounts of monoclonal antibodies: anti-Stro-1, anti-CD90, anti-CD106, anti-CD105, anti-CD146, anti-CD166, anti-CD44, anti-CD19, anti-CD45 (Human Mesenchymal Stem Cell marker antibody panel, R&D System, Minneapolis, Minn., USA), anti-CD44 (PE mouse anti-human CD44, BD Bioscience, Franklin Lakes, N.J., USA), anti-CD73 (FITC mouse anti-human CD73, BD Bioscience), anti-CD31 (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD11b (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD79a (PE, mouse anti-human, Abcam, Cambridge, UK), anti-CD13 (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-HLA-DR (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD14 (FITC, mouse anti-human, Abcam, Cambridge, UK), anti-CD34 (PE, mouse anti-human, Abcam, Cambridge, UK). At least 10,000 gated events were analyzed by flow cytometry with CellquestPro software. Results are expressed in mean fluorescence intensity (MFI), and expressed as percentage of positive cells (threshold: 95% of isotype).

(23) Differentiation Capacity

(24) ASC and DF were tested at passage 4 in specific media to assess the capacity of differentiation toward osteogenic lineage. The differentiation was evaluated by Alizarin red staining after culturing the cell during 3 weeks in specific differentiation medium (proliferation medium supplemented with dexamethasone (1 μM), sodium ascorbate (50 μg/ml), and sodium dihydrophosphate (36 mg/ml) [Qu et al., In Vitro Cell Dev Biol Anim. 2007; 43:95-100]. Osteogenic differentiation was confirmed by staining for calcium phosphate with Alizarin red after formalin fixation. In addition, immunohistochemistry for osteocalcin was performed to confirm the bone phenotype.

(25) Impact of Oxygen Tension and Fetal Bovine Serum (FBS) on Cell Proliferation: EdU Assay

(26) Cell proliferation capacity was tested by direct DNA synthesis measurement by 5-ethynyl-2′-deoxyuridine incorporation using Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Life Technology, Waltham, Mass., USA). ASC (n=3) and DF (n=3) were seeded in 21.5 cm.sup.2 culture dishes at a density of 5000 cells/cm.sup.2, and cultured for 24 hours in 10% FBS, 21% O.sub.2. Cells proliferation was then stopped by replacing the proliferation medium by the same, without FBS, for 24 hours. The cells were finally placed for 48 hours in the specific conditions: 0.1% O.sub.2, 5% O.sub.2 and 21% O.sub.2 in proliferation medium supplemented with 1% FBS or 5% FBS and EdU (5-ethynyl-2′-deoxyuridine, a nucleoside analog of thymidine and incorporated into DNA during active DNA synthesis) was added. After revelation with Alexa Fluor® 488, positive cells were counted by flow cytometry (FACScan; BD Biosciences, San Jose, Calif.).

(27) Growth Factor Secretion Profile

(28) After trypsinization, cells (after passage 3) were counted and 5 progressive dilutions were obtained: 100% ASC+0% DF; 75% ASC+25% DF; 50% ASC+50% DF; 25% ASC+75% DF; and 0% ASC+100% DF, and seeded in 12-well culture plates with cells at a density leading to about 80% to 95% confluence in triplicate for incubation in hypoxic chambers (Modular Incubator Chamber MIC-101; Billups-Rothenberg, Del Mar, Calif., USA) at 0.1% O.sub.2 and 5% O.sub.2, corresponding to highly hypoxic environment and tissular oxygen tension, respectively. The cells were exposed (for each dilution and oxygen tension) to normoglycaemic (1 g/L) or hyperglycaemic (4.5 g/L) proliferation media. After incubation for 24 hours in these controlled conditions; cell culture supernatants were harvested individually and stored at −20° C. for further growth factor quantification by enzyme-linked immunosorbent assay (VEGF, HGF, IGF-1, SDF-1α and basic FGF by Quantikine ELISA kit; R&D System, Minneapolis, Minn., USA). Cellular viability was assessed immediately after the hypoxic stress by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium solution (MTS; Promega, Leiden, the Netherlands) assay. Hypoxic/glycaemic stress tests and growth factor quantifications were performed in triplicate and duplicate, respectively. Results are expressed in picograms per millimeter.

(29) Statistical Analysis

(30) The one-sample Kolmogorov test and Q-Q plots were used to assess the normal distribution of values. Statistically significant differences between groups (with normal distribution) were tested by paired t-test and one-way analysis of variance with the Bonferroni post hoc test. Statistical tests were performed with PASW 18 (SPSS; IBM, New York, N.Y., USA); p<0.05 was considered significant.

(31) Results

(32) Surface marker profiles do not allow the distinction between the two cell populations (Table 2).

(33) TABLE-US-00002 TABLE 2 Surface marker characterization of human ASC and DF ASC DF % of positive cells % of positive cells Mesenchymal (stromal) cells markers CD13 99.06 99.86 CD44 95.53 99.97 CD73 93.78 99.86 CD90 98.63 100.00 CD105 96.86 99.78 CD166 60.74 96.51 Bone marrow-derived MSC markers CD106 5.41 2.83 Stro-1 4.03 5.73 CD146 7.16 33.91 Endothelial cells markers CD31 5.59 5.41 Hematopoietic lineage markers CD14 6.75 28.27 CD45 5.15 0.62 CD11b 5.80 8.65 CD34 5.53 0.54 Human leukocyte antigens HLA-DR 6.52 1.65 CD19 4.51 2.05 CD79α 5.10 0.37

(34) ASC and DF were positive (>90% of positive cells) for mesenchymal cell markers (CD13, CD44, CD73, CD90, CD105, CD166), negative for endothelial (CD31), bone-marrow-derived stromal cells (CD106, Stro-1, CD146) and hematopoietic markers (CD14, CD45, CD11b, CD34), and for HLA-DR, CD79α and CD19. After culture in specific differentiation media (FIG. 1), osteogenic differentiation capacity was demonstrated for both ASC and DF by Alizarin red staining and osteocalcin immunohistochemistry.

(35) ASC and DF had similar proliferation profile until passage 15 (FIG. 2, NS).

(36) ASC and DF viability was not significantly impacted after 24 hours of culture at 0.1% O.sub.2 and 5% O.sub.2 without FBS (FIG. 3). At 5% O.sub.2, DF viability was reduced when compared to ASC (87.04% of ASC survival, p<0.05).

(37) The study of HGF, IGF-1, bFGF and KGF secretion (at 0.1% and 5% O.sub.2, 4.5 g/l glucose) from the sequential dilutions of ASC and DF did not demonstrate any significant curve (FIG. 4).

(38) However, for VEGF and SDF-1α, linear regressions following ASC “contamination” by DF were observed. Indeed, SDF-1α secretion level decreases with increasing ASC proportion. This result is found in different conditions of oxygen tension (21%, 5% or 0.1%) or of glucose concentrations (1 g/l or 4.5 g/l) (FIGS. 5 to 10). Moreover, in high glucose culture conditions and at 0.1% O.sub.2 and 5% O.sub.2 VEGF secretion level increases with increasing ASC proportion (FIGS. 5A and 6A). The same measurements in low glucose conditions demonstrated significant linear regressions for VEGF secretion at 5% O.sub.2 (FIG. 8A).

(39) The relations were inversed since DF release higher levels of SDF-1α and VEGF was produced in higher rates by ASC, allowing the measurement of the cell proportion (ASC purity).