Integrin α10β1-selected Equine MSCs have Improved ...±10β1-selected-equine... · Citation:...

Somato Publications

Annals of Stem Cell Research

Annals of Stem Cell Research© 2018 Somato Publications. All rights reserved. 01 Volume 2 Issue 1 - 1003

Research Article

Integrin α10β1-selected Equine MSCs have Improved Chondrogenic Differentiation, Immunomodulatory and

Cartilage Adhesion CapacityKristina Uvebrant1, Linda Reimer Rasmusson1, Jan F. Talts1, Paolo Alberton2, Attila Aszodi2 and Evy

Lundgren-Åkerlund1*1Xintela AB, MediconVillage, Lund, Sweden

2Laboratory of Experimental Surgery and Regenerative Medicine, Clinic for General, Trauma and Reconstructive Surgery, Ludwig-Maximilians University, Munich, Germany

*Address for Correspondence: Evy Lundgren-Åkerlund, Xintela AB, Medicon Village, 223 63 Lund, Sweden, E-mail: [email protected]

Received: 19 December 2018; Accepted: 07 January 2019; Published: 09 January 2019

Citation of this article: Uvebrant, K., Reimer Rasmusson, L., Talts, JF., Alberton, P., Aszodi, A., Lundgren-Åkerlund, E. (2019) Integrin α10β1-selected Equine MSCs have Improved Chondrogenic Differentiation, Immunomodulatory and Cartilage Adhesion

Capacity. Ann Stem Cell Res, 2(1): 001-009.

Copyright: © 2019 Uvebrant K, et al. This is an open access article distributed under the Creative Commons Attribution License,

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACTCell therapy based on multipotent, adult mesenchymal stem cells (MSCs) is a promising method for the regeneration of cartilage tissue and treatment of osteoarthritis in both humans and animals. For safe and effective use of MSCs as therapeutic agents, quality control of the isolation and expansion methods as well as the final MSC product is of key importance. The aim of this study was to evaluate integrin α10β1 as a novel MSC biomarker to attest identity, purity and potency of MSC preparations. We found that MSCs, isolated from equine adipose tissue (AT) and selected using an antibody directed to integrin α10, expressed the stem cell markers CD44, CD90 and CD105, differentiated to chondrocytes, osteocytes and adipocytes and demonstrated immunomodulatory capacity as judged by suppression of T-cell proliferation and expression of PGE2. In addition, we found that integrin α10-selected equine AT-MSCs showed higher chondrogenic differentiation capacity, in pellet mass cultures, compared to unselected MSCs. Moreover, the integrin α10-selected fraction of MSCs showed significantly higher adhesion capacity to cartilage defects in ex vivo osteochondral explants compared to unselected MSCs demonstrating a higher ability of the α10-enriched MSCs to home to an osteochondral damage. Taken together, the results show that integrin α10β1 can identify a homogenous population of multipotent equine AT-MSCs with a high chondrogenic differentiation and immunomodulatory potency as well as homing capacity. Thus, we conclude that integrin α10β1 is a biomarker that can ensure quality, potency and consistency of MSC preparations for cartilage repair.

Keywords: Integrin α10β1, MSCs, Selection, Chondrogenesis, Immunomodulation

IntroductionArticular cartilage is highly prone to injury and pathological

degeneration, and once damaged, it has poor ability to regenerate and heal. Cartilage damage often leads to degradation of the cartilage extracellular matrix and the breakdown products can initiate catabolic gene expression and inflammation resulting in further degradation and inflammation, thus creating a vicious circle and

development of osteoarthritis (OA) [1]. There is a well-known link between early cartilage traumas, for example from sports injuries, and later development of OA [2].

Horses, as humans, frequently suffer from naturally occurring orthopedic disorders of which OA and joint cartilage injuries represent a substantial part [3]. The most common medical treatments for equine OA is intra-articular injection of corticosteroids, alone or in

Citation: Uvebrant, K., Reimer Rasmusson, L., Talts, JF., Alberton, P., Aszodi, A., Lundgren-Åkerlund, E., et al. (2019) Integrin α10β1-selected Equine MSCs have Improved Chondrogenic Differentiation, Immunomodulatory and Cartilage Adhesion

Capacity. Ann Stem Cell Res 2(1): 001-005.

Annals of Stem Cell Research© 2018 Somato Publications. All rights reserved. 02 Volume Issue 1 - 1003

combination with Na-hyaluronate, as well as application of systemic non-steroidal anti-inflammatory drugs (NSAIDs) [4]. These and other treatment regimens may only relieve symptoms and improve clinical recovery without regenerating the cartilage damage of the affected joint. Thus, there is to date no disease-modifying treatment available for OA in horses or in humans.

A growing body of evidence suggests that cell therapy using adult tissue-derived, multipotent mesenchymal stem cells (MSCs) is a promising method for tissue regeneration. Several studies in animals and humans have shown safety and efficacy of MSCs in treatment of various conditions, including cartilage damage [5,6]. The therapeutic effect of MSCs is believed to be multifactorial. MSCs can differentiate into the specific cell type of the tissue to be regenerated, and they can also modify the microenvironment by producing extracellular matrix, and releasing cytokines and growth factors as well as recruit and stimulate endogenous stem or progenitor cells. In addition, MSCs may also act therapeutically through their immunomodulatory properties [7]. Implantation of MSCs for cartilage repair and regeneration might therefore be a promising strategy given the potential of these cells to initiate and be part of tissue repair functionalities [5].

MSC preparations, however, are known to be heterogeneous, consisting of mixtures of cell types with varying capacities of differ-entiation and biological activity and, in addition, the heterogeneity varies from donor to donor [8]. Therefore, characterization of MSCs by quality control biomarkers is crucial not only to ensure relevant biological activities of the cells, but also for their safe and effective use as therapeutic agents. The International Society for Cellular Therapy (ISCT) have proposed a range of characteristics to define an MSC. For human MSCs, the minimal criteria defining these cells in vitro in-clude plastic adherence, trilineage (adipogenic, chondrogenic and os-teogenic) differentiation potential, and presence of the surface mark-ers CD73, CD90, CD105 as well as absence of CD45, CD34, CD14, CD11b, CD79α, CD19 and HLA-DR [9]. In a recent study on equine MSCs, although being plastic adherent and capable of trilineage dif-ferentiation, the cells could not meet the minimal criteria for CD markers required for human MSCs. Moreover, plastic adherence as a selection method for equine MSCs did not lead to uniform popula-tions [10] demonstrating the importance of using specific biomarker to identify MSCs.

It is now evident that MSCs selected by the ISCT criteria are heterogeneous, which may negatively affect their quality for use in stem cell therapy including cartilage repair. A solution to the heterogeneity problem could be to use more selective biomarkers to better define and isolate functional MSC populations [11]. In the present study, we evaluated the potential of integrin α10β1 as a novel cell surface biomarker on equine MSCs to determine identity, purity and potency of MSCs and thus to quality assure MSCs for cell therapy of damaged cartilage.

Integrin α10β1 was originally discovered as a major collagen-binding integrin on chondrocytes [12] and shown to be highly expressed in cartilage, both during development and in adult cartilage tissues [13]. In vitro and in vivo studies have recognized integrin α10β1 as an unique phenotypic marker for chondrocyte differentiation and a crucial mediator of cell-matrix interactions required for proper cartilage development [14]. Integrin α10 knockout mice have defects in the cartilaginous growth plate and, as a consequence, develop growth retardation of the long bones [15]. This was supported by a

more recent study showing that a naturally occurring mutation in the canine α10 integrin gene is responsible for chondrodysplasia in hunting dog breeds [16]. Furthermore, we have previously shown that integrin α10β1 is also present on bone marrow-derived human MSCs and that fibroblast growth factor 2 (FGF-2) supplementation in monolayer culture upregulates integrin α10, which in turn improves chondrogenic potential of the MSCs [17].

Here, we demonstrate that integrin α10β1 is present on a fraction of equine adipose tissue-derived MSCs (AT-MSC) and that integrin α10-selected MSCs have improved chondrogenic differentiation and immunomodulatory capacity as well as increased homing ability to damaged cartilage. Our results indicate that integrin α10β1 is a novel cellular biomarker for quality assurance of MSCs in cell therapy of cartilage damage including osteoarthritis.

Materials and Methods

Cell isolation and expansion

Equine adipose tissue (AT) was obtained from healthy equine cadavers with permit from the Swedish Board of Agriculture. Adipose tissue was aseptically minced into smaller pieces and washed in phosphate buffered saline (PBS) prior to adding an equal volume of 0.1% type I collagenase (Sigma-Aldrich) in PBS. The adipose tissue was then digested for 1-1.5h at 37ºC with gentle agitation until a smooth appearance. The digested samples were centrifuged twice for 5 min at 300xg followed by aspiration of the adipocyte layer and the collagenase supernatant. Remaining stromal vascular fraction was washed with DMEM/F-12 medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12) (Gibco), filtered through a 100 µm cell strainer and seeded into cell culture flasks containing DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) (Biological Industries) and 1x Antibiotic-Antimycotic (Gibco) at a plating density of approximately 35 ml adipose tissue per 200 cm2. Cells were incubated at 37ºC in a humidified hypoxia incubator with 4% O2 and 5% CO2 for 24h, then the non-adherent cells were discarded and media changed to culture medium consisting of DMEM/ F-12 supplemented with 5% platelet lysate (PL) from Cook Regentec, 50 ng/ml human fibroblast growth factor-2 (FGF-2) (Miltenyi) and Antibiotic-Antimycotic. The culture medium was replaced every 2-3 days and at 80% confluence, after 4-6 days, the AT-MSCs were harvested using Accutase (StemPro). Usually, 7-9 million MSCs per 35 ml adipose tissue seeded could be harvested. The MSCs were then reseeded for expansion at a cell density of 5000 cells/cm2 in culture medium.

At each passage the equine AT-MSCs were immunophenotyped by flow cytometry analysis using a panel of equine cell surface markers: integrin α10 (Xintela), CD90 (BD Pharmingen), CD44 (AdBSerotec), CD105 (AdBSerotec), CD45 (AdBSerotec) and MHC class II (AdBSerotec). In brief, 100 000 cells were washed in PBS supplemented with 1% FBS followed by incubation with the cell marker specific, fluorochrome-conjugated antibody. After one hour of incubation, cells were washed twice in buffer and analyzed using a BD Accuri C6 flow cytometer (BD Biosciences). Results were analyzed with BD Accuri C6 software and are expressed as frequency of positive cells.

Cell sorting and integrin α10 immunostaining

Equine AT-derived MSCs were cultured and expanded to passage 3 before subjected to integrin α10 fluorescence-activated cell sorting




by FACSAria (BD Biosciences). The cells were stained using a monoclonal integrin α10 antibody (Xintela) and live cells were sorted into two fractions: integrin α10high and integrin α10low. Discrimination of live/dead cells was assessed by 7-AAD staining (BioLegend). Sorted cells were washed in culture medium and re-seeded for recovery and expansion for one more passage before subsequent experiments.

Integrin α10-selected cells (α10high) were seeded in 8-well slide (ibidi) in culture medium for confocal microscopy analysis. After 48h of culturing, cells were washed and blocked in PBS with 2% BSA before incubation with the anti-integrin α10 antibody. As secondary antibody a goat-anti-mouse Alexa Fluor 488-conjugated antibody (Jackson Immunoresearch) was used and the stained cells were visualized by confocal microscope (LSM 800, Zeiss).

Chondrogenic differentiation

To determine the optimal chondrogenic differentiation condition for equine AT-MSCs, various growth factor concentrations and combinations of these were tested. Equine AT-MSCs were expanded to passage 2 and 200 000 cells were pelleted in 15 ml polypropylene tubes in basal chondrogenic differentiation medium with and without growth factors. The chondrogenic basal medium consisted of high glucose DMEM (Gibco) supplemented with 50 ug/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 1% Insulin-Transferrin-Selenium (Sigma-Aldrich) and 100 nM dexamethasone (Sigma-Aldrich). The different growth factors concentrations and combinations used were 10 ng/ml transforming growth factor β3 (TGFβ3) (Sigma-Aldrich), 10 ng/ml TGFβ3 and bone morphogenic protein 6 (BMP-6) (Sigma-Aldrich) or 20 ng/ml TGFβ3 and BMP-6. The MSC pellets were differentiated under hypoxia (4%) for 28 days or as indicated in the chondrogenic time study. Medium was changed three times per week. In the time study and when determination of chondrogenic differentiation capacity of non-selected and integrin α10-selected cells were conducted, 20 ng/ml TGFβ3 and BMP-6 was used.

Pellets were either embedded in Optimal Cutting Temperature compound (HistoLab) for cryosectioning or snap frozen in QIAzol (QIAGEN) and stored at -80ºC for RNA extraction. Non-induced chondrogenic pellets cultured in chondrogenic basal medium were used as negative controls.

For immunohistochemistry, the pellets were cryosectioned, fixed in acetone and subjected to immunostaining using antibodies directed to the cartilage specific proteins collagen type II (Abcam) and integrin α10 (Xintela). Sections were counterstained with hematoxylin and visualized in microscope (Leica LM R).

Adipogenic differentiation

To determine the adipogenic differentiation capacity, integrin α10-selected equine AT-MSCs, at passage 4, were added to a 12-well plate (38 000 cells/well) and allowed to attach in StemMACS MSC expansion media (Miltenyi) with 50 ng/ml FGF-2 and 1x Antibiotic-Antimycotic for 24h. This was followed by change to adipogenic medium consisting of DMEM/F12 (Gibco) supplemented with 15% rabbit serum (Sigma-Aldrich), 1 µM dexamethasone (Sigma-Aldrich), 100 µM indomethacin (Sigma-Aldrich), 500 µM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), and 1x Antibiotic-Antimycotic. Cells were cultured under hypoxic (4%) condition. Medium was changed every second day and maintained until fat droplets could be detected, usually within 2-3 days after adipocyte differentiation induction.

For analysis of adipocyte differentiation, cells were fixed in 10% formalin (Sigma-Aldrich) for 45 min, incubated in 60% isopropanol (Sigma-Aldrich) for 5 min and finally stained with 0.3% oil red O (Sigma-Aldrich) for 5 min. After subsequent aspiration and washing, cells were visualized under a bright field microscope. Non-induced adipogenic cells, cultured in StemMACS, were used as negative controls.

Osteogenic differentiation

For osteogenic differentiation the StemPro Osteogenesis differentiation kit (Gibco) was used according to the manufacturer´s protocol. In short, integrin α10-selected equine AT-MSCs were seeded in a 12-well plate (19 000 cells/well) and cultured in StemMACS MSC expansion media (Miltenyi) with 50 ng/ml FGF-2 and 1x Antibiotic-Antimycotic for 24h before start of differentiation with osteogenic induction medium. Cells were cultured in hypoxia (4%) and the medium was changed every 2-3 days. After 21 days, cells were fixed with 4% formaldehyde and rinsed with PBS before staining with Alizarin Red solution (Millipore) for 3 min followed by washing with PBS. Osteogenic differentiation was visualized under light microscope. Non-induced osteogenic cells, cultured in StemMACS, were used as negative controls.

Real-time PCR analysis

Total RNA was extracted by homogenization of pellets in QIAzol (QIAGEN) using Precellys lysing kit beads and homogenizer (Bertin Instruments) followed by RNA isolation according to the RNeasy Lipid Tissue Mini kit protocol (QIAGEN). The RNA was reverse-transcribed into cDNA using SuperScript VILO kit (Invitrogen) according to the manufacturer´s protocol. Real-time PCR was conducted using TaqMan assays (Applied Biosystems) for the following equine gene transcripts: GAPDH, COL2A1, COL1A1 and ACAN (glyceraldehyde 3-phosphate dehydrogenase, collagen type II, collagen type I and aggrecan, respectively). TaqMan Universal Master Mix II (Applied Biosystems), TaqMan primers and cDNA were mixed according to the manufacturer’s instructions and run in a StepOne Plus Real Time PCR System device (Applied Biosystems). The relative mRNA expression was calculated using the ∆∆Ct method where GAPDH was the housekeeping control (∆Ct) and normalized towards unsorted cells (2 –ΔΔCt)[18].

Immunomodulation assay

The immunomodulatory capacity of integrin α10-selected equine AT-MSCs was assessed in a T-cell proliferation assay. Equine peripheral blood mononuclear cells (PBMC) (Cellider Biotech) were stimulated with 2.5 μg/ml Concanavalin A (Con A) in the presence of different ratios of MSC to PBMC (1:1, 1:5 and 1:10). All cells were incubated in RPMI 1640 (Gibco) with 10% FBS (Biological Industries), 10 mM HEPES (Gibco) and Antibiotic-Antimycotic for 72h. Bromodeoxyuridine (BrdU) was added to the cell cultures 24h before analysis. Staining of the cells was conducted according to the BrdU flow kit manual (BD Pharmingen) and analyzed in an Accuri C6 flow cytometer (BD). FITC-conjugated horse CD4 antibody (Bio-Rad) was used to cell surface stain the equine CD4 expressing cells. The frequencies of CD4 and BrdU positive cells were used as parameters for quantification of T-cell proliferation.




Pro-inflammatory stimulation assay

Equine AT-MSCs at passage 3 were seeded in a 6-well plate (8000 cells/cm2) in StemMACS supplemented with 50 ng/ml FGF-2 and 1x Antibiotic-Antimycotic and cultured over night. MSCs were then exposed to recombinant equine interferon- γ (IFN-γ) (R&D Systems) and recombinant equine tumor necrosis factor-α (TNF-α) (R&D Systems) at 20 ng/ml for 72h and supernatants were collected and stored at -80°C until assayed. Basal medium without inflammatory factors was used as a negative control and the cells were cultured in hypoxia (4%). Prostaglandin E2 (PGE2) levels in supernatant from equine MSCs cultured with or without IFN-γ and TNF-α were determined with the Prostaglandin E2 Parameter Assay Kit (R&D Systems) according to manufacturer´s instructions. Samples were read using a Spectra MAX i3 (Molecular Devices) at 450 nm and 570 nm, and quantified against a standard curve.

Ex vivo cell binding assay

Femoral condyles from 1-1.5 years old bulls were obtained from a local slaughterhouse (permission number: 1069/2009). Osteochondral explants were isolated using a 7 mm trephine drill (Hager and Meisinger GmbH). Biopsies were sawed to about 5 mm in height carrying the full thickness articular cartilage (circa 1 mm) and the underlying subchondral bone (4 mm), and washed in PBS supplemented with 1x Antibiotic-Antimycotic. One mm dermatological curette (PFM Medical) was used to create longitudinal chondral and subchondral defects on the articular cartilage. After a further wash in PBS Antibiotic-Antimycotic, explants were moved to a bacterial Petri dish and covered with DMEM/F12 supplemented with 5% PL and Antibiotic-Antimycotic (incubation medium). To avoid binding of cells to areas outside of the articular surface, biopsies were briefly dipped in melted paraffin along their whole length till the cartilage layer, and immediately attached to the cap of 2 ml cryovials (Thermo Scientific). Cryovials with osteochondral plugs were left standing up-side-down with 800 µl incubation medium until the assay started.

Equine AT-MSCs were collected and counted as described above. Aliquots of 5x104 cells per explant were fluorescently labelled with 10 µM live cell tracer Vybrant CFDA-SE (Invitrogen) for 15 min, washed in PBS, resuspended in 500 µl incubation medium and added to the cryovials containing the explants resulting in a final volume of 1.3 ml. Dynamic cell binding was performed at 37°C using a Multi-Rotator (Grant Instruments LTD) which was set to execute five alternated clockwise/counterclockwise complete turns at 7 rpm for 1h. Afterwards, the suspensions were discarded and the cryovials rinsed with PBS. Unadhered cells were removed by washing explants in PBS twice for 2 min on the Multi-Rotator with the same conditions as described above. Adhered cells on the chondral and subchondral defect area were visualized with the fluorescence microscope Axio Observer Z1 (Zeiss) using the GFP filter, counted with the counting plug-in of Image J software (http://rsb.info.nih.gov/ij/), and reported as adhered cells per mm². Cell binding was assayed using one AT-MSC donor, three independent times in triplicate. Data represents the mean between the three independent runs ± standard deviations.

Statistical analysis

Data are expressed as the mean ± standard deviation (SD). Significance of the experiments was assessed using unpaired Student´s

t test. The level of significance was set at p<0.05 (*), p<0.01 (**) and p<0.001 (***).

Results

Expression of stem cell markers on equine AT-MSCs

To demonstrate expression of integrin α10β1 (integrin α10) and other stem cell markers on equine adipose tissue-derived MSCs (AT-MSCs), cells were isolated from neck adipose tissue and expanded in DMEM/F-12 medium containing 5% PL and 50 ng/ml FGF-2 under hypoxic (4%) conditions. As shown in Figure 1A, the MSCs exhibited typical phenotype with high expression levels of the commonly used stem cell markers CD44, CD90 and CD105 and no expression of the leukocyte marker CD45 or of MHC class II. Expression of integrin α10β1, on the other hand, was detected in a subfraction of the MSCs with an expression range of 15-60% among different donors after the first passage. Average expression levels from 5 different AT-MSC donors are presented in Figure 1B.

Optimization of chondrogenic differentiation

To investigate and optimize the differentiation of equine AT-MSCs to chondrocytes, we compared the effect of TGFβ3 alone to the effect of combinations of BMP-6 and TGFβ3 in promoting chondrogenic differentiation. Equine AT-MSCs expanded in PL and FGF-2 for 2 passages were subsequently cultured in pellet mass for 28 days either in the absence of growth factors or in the presence of 10 ng/ml TGFβ3 only, or in the presence of TGFβ3 combined with BMP-6 at 10 or 20 ng/ml. Expression of collagen type II and integrin α10β1

Figure 1: Flow cytometry analysis of integrin α10 and other stem cell markers on equine AT-MSCs. (A) Representative flow cytometry profiles of equine AT-MSCs at passage 3 with expression levels of the stem cell markers CD44, CD90, and CD105 as well as integrin α10, and the hematopoietic cell marker CD45 and MHC class II. The red curves show the binding of antibodies and the black curves (negative control) represent MSCs in the absence of antibodies. (B) Mean percentage expression values of the different cell markers ± SD on MSCs from 5 different donors.




was analyzed on cryosections of the pellets by immunohistochemistry. We found that a combination of BMP-6 and TGFβ3 was necessary to promote chondrogenic differentiation of the equine AT-MSCs and that 20 ng/ml of TGFβ3 and BMP-6 was more effective than 10 ng/ml (Figure 2A).

We also investigated the correlation between the expression of integrin α10 and collagen type II by immunohistochemical analysis of pellet cryosections at different time points (7, 14, 21, and 28 days) during the differentiation process. After 14 days, cells in the outermost layer of the pellet started to produce collagen type II. After 21 days the expression was more pronounced and widespread demonstrating a transition of MSCs to chondrocytes. After 28 days, expression of collagen II was further increased and was more uniform in the pellet. Integrin α10 was detected already at 7 days and its expression increased with time of differentiation (Figure 2B).

Integrin α10-selected equine AT-MSCs have improved chondrogenic differentiation capacity

MSC preparations are known to be heterogeneous and since we have found that expression of integrin α10 on MSCs varies between donors, we sorted the integrin α10-expressing MSCs by FACS using an integrin α10-specific monoclonal antibody (Figure 3A). In order to further verify the expression of integrin α10 on AT-MSCs, α10 sorted cells were plated and cultured on a glass chamber slide. Immunofluorescence staining and confocal microscopy analysis demonstrated surface localization of α10 integrin on equine MSCs (Figure 3B).

Next we investigated the capacity of the selected MSCs to differentiate to chondrocytes in comparison with unselected cells. The two cell fractions, integrin α10 high (α10high) and integrin α10 low (α10low), as well as unsorted cells, were expanded in AT-MSC culture medium containing PL and FGF-2, and then transferred to pellet mass cultures in chondrogenic basal medium in the presence of 20 ng/ml TGFβ3 and BMP-6 for chondrogenic differentiation. After 28 days, pellets were cryosectioned and analyzed by immunohistochemistry for expression of collagen type II. Representative images (from one

of four different donors) show high expression of collagen type II in the pellets from α10high MSCs, low collagen type II expression in the α10low pellets and intermediate levels when the pellets were made from unselected MSCs (Figure 3C). These results demonstrate a correlation between integrin α10β1 expression level and chondrogenic differentiation capacity of equine MSCs.

To further investigate this correlation, mRNA expression levels of collagen II, aggrecan and collagen I, in pellets from the α10high

and α10low MSC fractions as well as the unselected MSCs, were analyzed by quantitative RT-PCR from 2 different donors. GAPDH was used as housekeeping control and unselected cells were used as reference for ΔΔCt calculation (Figure 3D). The results showed higher gene expression of collagen type II (COL2A1) and aggrecan (ACAN) and lower expression of collagen type I (COL1A1) in α10high pellets compared to unselected and α10low pellets, verifying improved chondrogenic differentiation capacity of integrin α10-selected MSCs.

Integrin α10-selected equine AT-MSCs have trilineage differentiation capacity

In order to further understand the differentiation potential of the integrin α10-selected equine AT-MSCs, we investigated their osteogenic and adipogenic differentiation capacity. We found that the integrin α10-selected MSCs were able to differentiate, in addition

Figure 2: Optimization of the chondrogenic differentiation of equine AT-MSCs. (A) Optimal differentiation conditions were achieved with a combination of TGFβ3 and BMP-6 at 20 ng/ml as indicated by collagen II and integrin α10 staining. (B) Time study of chondrogenic differentiation show pronounced expression of type II collagen and integrin α10 after 3 and 4 weeks in culture.

Figure 3: Selection and chondrogenic differentiation of integrin α10high equine AT-MSCs. (A) Equine AT-MSCs were sorted by FACS into two cell fractions: integrin α10high (α10high) and integrin α10low (α10low). The figure show typical gating of the integrin α10-expressing MSCs in a sorting experiment. (B) Immunofluorescence staining with an integrin α10 antibody and confocal microscopy show the surface localization (green) of integrin α10 on monolayer cultured α10high AT-MSCs. Nuclei were visualized by DAPI staining (blue). (C) Pellets mass cultures from unselected, integrin α10high and integrin α10low MSCs were cryosectioned and analyzed by immunohistochemistry using antibody against collagen type II. The images show results from one of four different donors with similar results. Results show that α10high MSCs have improved expression of collagen II. (D) Real-time PCR analysis of pellet mass cultures show higher expression of type II collagen and aggrecan in pellets with α10high MSCs, and higher expression of collagen type I in pellets with α10low MSCs. The results are from one representative experiment from two different donors performed in triplicate and presented as mean relative gene expression ± SD.




to chondrocytes, into osteoblasts and adipocytes when incubated under the appropriate conditions, which demonstrate trilineage differentiation capacity of the integrin α10-selected MSCs (Figure 4).

Integrin α10-selected equine AT-MSCs demonstrate immunomodulatory capacity

To investigate if integrin α10high equine AT-MSCs have immunomodulatory effect, the selected MSCs from two different donors were co-cultured with Con A-stimulated PBMCs at different ratios, and the effect on T-cell proliferation was analyzed. The results show a significant decrease in proliferating CD4 positive T-cells with increasing number of α10high equine AT-MSCs, with the largest immunosuppressive effect at one MSC to one PBMC ratio (Figure 5A).

To further investigate the immunomodulatory properties of the integrin α10-selected equine MSCs, the MSCs were stimulated with TNF-α and IFN-γ, cultured under hypoxic conditions for 72h and PGE2 levels were then analyzed by ELISA. The results show higher levels of PGE2 in supernatants from stimulated cells relative to unstimulated cells after TNF-α and IFN-γ stimulation. In addition, we found that the PGE2 levels were significantly higher in the integrin α10-selected (α10high) MSCs culture compared to unselected MSCs culture after stimulation (Figure 5B).

Integrin α10-selected equine AT-MSCs have improved capacity to adhere to cartilage defects

In order to evaluate the potential role of integrin α10β1 as a cartilage homing receptor on MSCs, we performed an ex vivo adhesion assay using cartilage explants from bovine femoral condyles with artificial chondral and subchondral damages (Figure 6A and 6B). Equine AT-MSCs (from one donor) sorted into integrin α10high, integrin α10low as well as unsorted MSCs were fluorescently-labelled with CFDA-SE and were allowed to adhere to the cartilage explants for 1h. The adhesion was performed under dynamic conditions to avoid non-specific binding of the cells, and MSC attachment was analyzed by fluorescent microscopy. We found significantly higher number of bound integrin α10high MSCs to both the chondral (Figure 6C) and the subchondral (Figure 6D) damaged cartilage compared to unselected MSCs showing that integrin α10-selected MSCs have improved capacity to home and adhere to cartilage defects.

DiscussionMSCs, isolated from various adult tissues, are attractive cells to

use in regenerative medicine due to their relatively easy accessibility, large potential for in vitro expansion and safety issues [5,6]. However, evidence has been gathered suggesting that implantation of various MSC preparations could pose risks of immune reactions, and growth of unwanted tissues [19] owing to heterogeneity of the stem cell preparations [8]. Therefore, a critical issue in cell therapy is to ascertain that the cells are indeed MSCs (identity), the MSCs preparation is homogenous (purity) and that they have the capacity to regenerate the damaged tissue (potency). Since the ISCT-proposed markers for stem cell identification were published in 2006, several other MSC markers have become widely accepted [20]. No markers have been identified as exclusively expressed by MSCs, but the growing panel of MSC-associated markers increases the validity of the identification of isolated MSCs by the original ISCT-proposed markers [21]. Although these surface markers can identify MSCs,

Figure 4: Osteogenic and adipogenic differentiation of integrin α10-selected equine MSCs. The capacity of integrin α10-selected MSCs to differentiate to osteoblasts and adipocytes was shown by Alizarin red and Oil red O staining, respectively.

Figure 5: Immunomodulation by integrin α10-selected equine AT-MSCs. (A) The immunosuppressive activity of integrin α10-selected MSCs were demonstrated by the decrease in T-cell proliferation in the presence of selected MSCs. The results show proliferation measured as frequency of BrdU and CD4 positive cells. The experiment was performed with MSCs from two different donors in triplicates and the results are mean values ± SD. *and ***indicates statistically significant difference, *p<0.05, ***p<0.001. (B) PGE2 levels in supernatants from unstimulated MSC and pro-inflammatory cytokine stimulated MSC. The PGE2 levels were higher in the integrin α10high selected MSCs compared to unselected MSCs especially in the stimulated MSCs. The experiment was performed with MSCs from two different donors in triplicates and the results are mean values ± SD. **indicates statistically significant difference, p<0.01.




they cannot solve the problem with heterogeneous MSC preparations and inconsistency between different donors and different sources, and do not always correlate to function and potency [21,22]. Therefore, improved methods to generate homogenous, high quality MSCs will increase the regenerative capacity of isolated and expanded MSCs, and thus prove crucial for their safe and effective use as therapeutic agents. We show in this study that the cell surface marker integrin α10β1 is present on a subfraction of equine adipose tissue-derived MSCs, and that integrin 10-selected equine AT-MSCs show improved chondrogenic and immunomodulatory capacity in vitro as well as increased adhesion to cartilage defects ex vivo.

Multipotent MSCs isolated from various adult tissues differ in their gene expression and phenotypic characteristics dependent on the tissue source and the culture conditions [22,23]. Specific cell surface markers, to select highly chondrogenic MSCs, is a sought-after tool in regenerative medicine aiming to repair cartilage defects. Earlier efforts to characterize and link cell surface biomarkers to subfractions of MSCs with increased chondrogenic capacity have previously concentrated on CD105 [24,25], CD146 [26,27] and stage-specific embryonic antigen-4 (SSEA-4) [28]. SSEA-4 is commonly used to characterize undifferentiated human embryonic stem cells, but it is also expressed on human MSCs from a variety of sources, including bone marrow. However, SSEA-4 levels in bone marrow-derived MSCs were unrelated to the chondrogenic potential in vitro [28]. CD146, on the other hand, was useful to isolate chondrogenic progenitor cells from late stage osteoarthritis cartilage [27]. Moreover, bone marrow derived MSCs enriched for high CD146 expression, heightened the relative glycosaminoglycan to DNA ratio when differentiated in chondrogenic media [26].The role of CD105 for chondrogenesis has

been examined in synovial membrane derived MSCs. These cells, when enriched for high CD105 expression, were shown to promote chondrogenesis in spheroid cultures [24]. In another study, a stronger chondrogenic potential of CD105 positive MSCs in comparison to that of CD105 negative MSCs could be shown, and this enhanced chondrogenesis was dependent on activation of the TGF-β/Smad2 signaling pathway [25].

We have previously demonstrated that integrin α10β1 is expressed by human MSCs isolated from bone marrow and that it can be upregulated by FGF-2 which correlates with improved chondrogenic potential in pellet mass cultures as shown by increased collagen type II and aggrecan expression [17]. In that study, the MSCs were differentiated in the presence of 10 ng/ml of TGFβ. In the present study we found that equine AT-MSCs did not differentiate to chondrocytes in the presence of 10 ng/ml TGFβ3. Instead, a combination of TGFβ3 and BMP-6 (20 ng/ml of each) was necessary to stimulate chondrogenic differentiation in pellet mass cultures. This is in agreement with a previous study with human AT-MSCs showing low chondrogenic capacity in the presence of TGF alone [29]. It has been suggested that AT-MSCs lack TGFβ-receptors making them unresponsive to a chondrogenic induction signal mediated by TGFβ [30]. In agreement with our finding, it has been reported that a deficiency in TGFβ-receptor expression can be overcome by the addition of BMP-6, which will induce TGFβ-receptor expression on human adipose tissue-derived MSCs [29].

Adipose tissue (AT) is an ideal source to obtain large amounts of MSCs known to have chondrogenic, osteogenic and adipogenic differentiation capacity [31]. In addition, MSCs from adipose tissue have no ethical issues, which is a regulatory advantage in the development of a cell therapy product. By flow cytometric characterization of equine AT-MSCs we could demonstrate expression of the commonly used markers CD44, CD90 and CD105, and lack of the leukocyte cell marker CD45 as well as MHC class II. Using an antibody specific for integrin α10, we identified a subfraction of equine AT-MSCs that expressed integrin α10 on the cell surface. We also found that the expression level of integrin α10 varied between donors although the expressions of the other tested markers were consistently high as judged by flow cytometry. Interestingly, we found that the integrin α10-selected (integrin α10high) equine AT-MSCs have improved chondrogenic differentiation capacity compared to unselected MSCs and MSCs with no or low integrin α10 expression (integrin α10low). This was demonstrated in pellet mass cultures both on protein and on mRNA levels. In all four tested donors, the chondrogenic capacity was consistently higher in the integrin α10high MSCs compared to unselected and integrin α10low

MSCs even though the degree of chondrogenesis in unselected and integrin α10low MSCs did slightly vary between the donors. We also found that the integrin α10-selected MSCs have immunomodulatory capacity as judged by suppression of T-cell proliferation. In addition, the selected MSCs showed increased expression of PGE2 compared to unselected MSCs. PGE2 has been suggested to play an important role in the immunomodulatory properties exhibited by MSCs and in the cross-talk between MSCs and macrophages which might be one of the mechanisms involved in tissue regeneration and repair [32,33]. Our results thus suggest that the cell surface marker integrin α10 identifies a homogenous subfraction of MSCs with high quality and potency properties for cell therapy of cartilage. Furthermore, selection of this subfraction provides a novel and promising way to equalize donor-

Figure 6: Increased adhesion of integrin α10-selected equine AT-MSCs to damaged osteochondral explants. Chondral and subchondral defects, generated in osteochondral explants, were used to determine adhesion of integrin α10high, integrin α10low and unselected equine AT-MSCs ex vivo. A top view (A) and a side view (B) of representative osteochondral plugs (AC-articular cartilage; SB-subchondral bone) show the generated chondral (c) and subchondral (sc) defects. Adhesion of CFDA-SE labelled MSCs to the chondral and subchondral defects under dynamic conditions was quantified after one hour of incubation using fluorescence microscopy. The bars show the adhesion of α10low and α10high MSCs per mm² relative to the adhesion of unselected MSCs to chondral (C) and subchondral defects (D).The integrin α10high MSCs demonstrated higher binding capacity compared to unsorted and α10low MSCs. The experiment was performed with MSCs from one donor and the results are from 3 independent binding assays performed in triplicates. Bars show mean ± SD. *indicates statistically significant difference, p<0.05.




to-donor identity and potency variations which is a largely unmet need in the regenerative medicine field [34]. Importantly, the potency of the integrin α10-selected equine AT-MSCs, used in the present study, has recently been verified in an equine in vivo model for post-traumatic OA (submitted for publication).

For optimal therapeutic effect, the MSCs should locate to the damaged area to exert their regenerative functionalities. Whether this therapeutic effect is dependent on differentiation and tissue integration of the MSCs, immunomodulation, modification of the local environment by activating and recruiting endogenous cells, or combinations of those, is however still not clear. Since integrin α10β1 is a collagen binding receptor, we hypothesized that integrin α10 will promote the MSCs to home and bind to cartilage damages with exposed collagen. To investigate the cartilage homing capacity of the integrin α10-selected equine AT-MSCs, we studied the adhesion of integrin α10high MSCs in comparison with integrin α10low MSCs and unselected MSCs to cartilage explants with a chondral or subchondral damage. The experiments were performed under dynamic conditions to better mimic homing in a non-static clinical situation and avoid unspecific binding. Our results indeed showed that integrin α10high MSCs adhered to chondral and subchondral defects to a higher degree compared to both unselected and integrin 10low MSCs. This indicates that MSCs expressing high levels of integrin α10β1 have an advantage in homing to the site where they should exert their effect. Further studies will investigate the role of integrin α10 in homing and engraftment of MSCs in an in vivo model.

ConclusionIn the present study, we demonstrate that integrin α10β1 is a

novel cell surface biomarker on equine AT-MSCs that can determine identity, homogeneity and potency of isolated and expanded MSCs. In addition, we show that integrin α10β1 can act as homing receptor to direct MSCs to damaged cartilage. Thus, our findings will have an important impact on the development of a safe and effective MSC therapy of degenerative joint disease including cartilage damage and osteoarthritis in horses as well as other animals and humans.

AcknowledgmentsThis study was partly supported by the DFG grant to AA and ELA

(AS 15/11-1) as a subproject of FOR 2407.

Conflict of Interest StatementEvy Lundgren-Åkerlund is a major shareholder in Xintela. Evy

Lundgren-Åkerlund, Kristina Uvebrant, Linda Reimer Rasmusson are employed by Xintela and Jan F. Talts is a former employee at Xintela.

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https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.2042-3292.2001.tb00082.x








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https://www.ncbi.nlm.nih.gov/pubmed/?term=The Necessity of a Systematic Approach for the Use of MSCs in the Clinical Setting





























https://www.ncbi.nlm.nih.gov/pubmed/?term=Canine chondrodysplasia caused by a truncating mutation in collagen-binding integrin alpha subunit 10


























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