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b Krembil Discovery Tower, 60 Leonard Ave, 5KD-415, Toronto, ON, M5T 2S8, Canada.
A. Gómez-Aristizábal
Footnotes
a AGA and AS contributed equally to this work. b Krembil Discovery Tower, 60 Leonard Ave, 5KD-415, Toronto, ON, M5T 2S8, Canada.
Affiliations
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, CanadaThe Arthritis Program, Toronto Western Hospital, Toronto, ON, Canada
b Krembil Discovery Tower, 60 Leonard Ave, 5KD-415, Toronto, ON, M5T 2S8, Canada.
A. Sharma
Footnotes
a AGA and AS contributed equally to this work. b Krembil Discovery Tower, 60 Leonard Ave, 5KD-415, Toronto, ON, M5T 2S8, Canada.
Affiliations
The Arthritis Program, Toronto Western Hospital, Toronto, ON, CanadaDivision of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada
c Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, ON, M5S 3G9, Canada.
M.A. Bakooshli
Footnotes
c Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, ON, M5S 3G9, Canada.
Affiliations
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, CanadaDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, ON, Canada
d Krembil Discovery Tower, 60 Leonard Ave, 5KD-413, Toronto, ON, M5T 2S8, Canada.
M. Kapoor
Footnotes
d Krembil Discovery Tower, 60 Leonard Ave, 5KD-413, Toronto, ON, M5T 2S8, Canada.
Affiliations
The Arthritis Program, Toronto Western Hospital, Toronto, ON, CanadaDivision of Genetics and Development, Krembil Research Institute, Toronto, ON, Canada
e Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Room 510, Toronto, ON, M5S 3E1, Canada.
P.M. Gilbert
Footnotes
e Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Room 510, Toronto, ON, M5S 3E1, Canada.
Affiliations
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, CanadaDonnelly Centre for Cellular and Biomolecular Research, University of Toronto, ON, CanadaDepartment of Biochemistry, University of Toronto, ON, Canada
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, CanadaThe Arthritis Program, Toronto Western Hospital, Toronto, ON, CanadaDivision of Genetics and Development, Krembil Research Institute, Toronto, ON, CanadaCell Therapy Program, University Health Network, Toronto, Canada
The Arthritis Program, Toronto Western Hospital, Toronto, ON, CanadaDivision of Orthopaedic Surgery, Toronto Western Hospital, University of Toronto, Toronto, ON, Canada
a AGA and AS contributed equally to this work. b Krembil Discovery Tower, 60 Leonard Ave, 5KD-415, Toronto, ON, M5T 2S8, Canada. c Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Toronto, ON, M5S 3G9, Canada. d Krembil Discovery Tower, 60 Leonard Ave, 5KD-413, Toronto, ON, M5T 2S8, Canada. e Donnelly Centre for Cellular and Biomolecular Research, 160 College Street, Room 510, Toronto, ON, M5S 3E1, Canada.
Although, mesenchymal stromal cells (MSCs) are being clinically investigated for their use in osteoarthritis (OA), it is unclear whether their postulated therapeutic properties are equally effective in the early- and late-stages of OA. In this study we investigated MSC cytokine secretion post-exposure to synovial fluid (SF), obtained from early- vs late-stage knee OA patients to justify a potential patient stratification strategy to maximize MSC-mediated treatment effects.
Method
Subjects were recruited and categorized into early- [Kellgren–Lawrence (KL) grade I/II, n = 12] and late-stage (KL-III/IV, n = 12) knee OA groups. SF samples were obtained, and their proteome was tested using multiplex assays, after 3-days culture, with and without MSCs. SFs cultured without MSCs were used as a baseline to identify MSC-secreted factors into SFs cultured with MSCs. Linear mixed-effect models and non-parametric tests were used to identify alterations in the MSC secretome during exposure to OA SF (3-days). MSCs cultured for 3-days in 0.5% fetal bovine serum (FBS)-supplemented medium were used to compare SF results with culture medium.
Results
Following exposure to OA SF, the MSC secretome contained proteins that are involved in tissue repair, angiogenesis, chemotaxis, matrix remodeling and the clotting process. However, chemokine (C-X-C motif) ligand-8 (CXCL8; chemoattractant), interleukin-6 (IL6) and chemokine (C-C motif) ligand 2 (CCL2) were elevated in the MSC-secretome in response to early- vs late-stage OA SF.
Conclusion
Early- vs late-stage OA SF samples elicit a differential MSC secretome response, arguing for stratification of OA patients to maximize MSC-mediated therapeutic effects.
Osteoarthritis (OA) management is limited to either symptom alleviation or arthroplasty, leaving the underlying cartilage, bone and synovium pathology unaddressed. Recently, mesenchymal stromal cells (MSCs) have emerged as a promising cell-based therapy for OA
. Intra-articular delivery of MSCs for OA treatment is being investigated in clinical trials, and safety, feasibility and preliminary efficacy of MSCs has been reported
. Questions remain about dose, carrier choice and whether MSCs are truly disease modifying.
MSCs have shown both reparative and anti-inflammatory effects in animal models of OA, with contribution to cartilage regeneration, increased systemic levels of interleukin-10 (IL10), and reduction of joint-specific prostaglandin-E2
. However, these effects have not been fully confirmed in clinical trials. Importantly, the MSC secretome is considered important in inducing anti-inflammatory and anti-catabolic effects in tissues
. Identifying and quantifying the MSC secretome in response to different OA joint microenvironments are therefore critical steps towards elucidating the mechanism of action of MSCs in OA.
Importantly, it is unclear whether MSC trophic factor secretion is differentially modulated in the context of an OA joint; specifically, in early- vs late-stage OA. Since MSC trophic factor secretion can facilitate cartilage repair and reduce inflammation, interrogating the MSC secretome after exposure to different OA microenvironments is necessary to identify OA patient subsets that would most benefit from MSC therapy. Identifying and targeting those patients at the outset will result in unequivocal clinical outcomes in late-stage clinical trials; a lesson learned from subset successes of MSCs in GvHD (graft vs host disease), where MSCs were effective in gastrointestinal GvHD but not skin GvHD
In this study, we systematically assess the MSC secretome after exposure to different synovial fluids (SFs) (cell depleted), from early- and late-stage OA patients, using multiplex immunoassays and report differential responses to OA SF stages.
Materials and methods
OA patients
To determine the impact of different OA microenvironments on the MSC secretome, subjects diagnosed with primary knee OA were studied. Grade I and II, defined by the Kellgren–Lawrence (KL) rating scale, were categorized as early-stage, while patients with KL-III/IV were categorized as late-stage. Samples were acquired from patients who provided written informed consent to participate in this study. The University Health Network Research Ethics Board approved the study and acquisition of human tissues (bone marrow acquired at Princess Margaret Hospital; Protocol ID: #06-446-CE, SF acquired at Toronto Western Hospital; #10-0455-AE). Patients were Caucasian, 30–65 years old and with 25–65 kg/m2 body mass index (BMI). They were symptomatic, with enough severity for the need of intervention, and were off of any intra-articular corticosteroid for at least 3-months before SF draw. Patients with a diagnosis other than primary OA were excluded. Three donor-MSCs were used for downstream validation studies to verify secreted cytokines identified in the screen, performed with a single donor-MSC (Fig. 1).
Fig. 1Experimental plan diagram: To identify the secretome of MSCs in OA SFs and possible differential effects of SFs from different OA stages, a single-donor MSC was cultured in OA SFs and later the conditioned SFs were tested using a 64-plex immunoassays, from which four cytokines were selected for further analysis with MSCs from three donors using a 4-plex immunoassay.
SF was aspirated under sterile conditions, centrifuged 10 min at 2348 ×g, aliquoted and stored at −80°C (this ensured lysis of any remaining cells in the SF). SF samples were subjected to a single freeze–thaw cycle.
MSC isolation, culture, and selection
MSCs were acquired from bone marrow aspirated from the iliac crest of healthy donors, and expanded (up to passage 3–4) as previously described
, using Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). MSCs were cryopreserved and thawed >3 days before starting an experiment. MSCs were characterized for cell surface molecules (Supplementary Table 1) and trilineage differentiation. All MSCs exhibited immunomodulatory properties as assayed by T cell proliferation inhibition and induction of M2-Monocyte polarization (Supplementary Fig. 1).
Cytokine analysis
For multiplex analysis, MSCs were cultured for 3 days in 90% SF and 10% DMEM at 71.4 × 103 cells/ml. Briefly, MSCs were added to SFs by “injecting” MSCs, suspended in DMEM, into wells containing SF and later centrifuging to allow distribution of MSC suspension throughout the SF. For MSC-only (i.e. no SF) secretome analyses, cells were cultured in DMEM containing 0.5% FBS. Interleukin-6 (IL6), chemokine (C-X-C motif) ligand-8 (CXCL8), chemokine (C-C motif) ligand-2 (CCL2) and chemokine (C-X-C motif) ligand-12 (CXCL12) were selected for analyses as a preliminary multiplex (64 cytokines) screening of a single donor-MSC (Fig. 1 and Supplementary Table 3) identified IL6 and CCL2 as the two most highly secreted cytokines; CXCL8 and CXCL12 were selected as they were differentially secreted by MSCs in early- vs late-stage OA SFs. Analyses were performed on three donor-MSCs and 12 early- and late-stage OA patient SFs; each using a Bio-Plex MAGPIX 4-plex immunoassay (Bio-Rad, Mississauga, ON), as per manufacturer's instructions. SF viscosity was reduced before immunoassays by 1-h incubating samples with 2 mg/mL hyaluronidase (Sigma–Aldrich, Oakville, ON) at room temperature. Neat OA SF cultured for 72 h in the absence of cells (i.e., ‘baseline’) vs MSC treatment were used to determine the MSC levels of cytokines (ΔSF: pg/mL). Changes in cytokine levels were solely attributed to MSC secretion as no other cells were present.
Statistical analysis
Descriptive statistics were obtained. Samples were analyzed by OA stage [early- (n = 12) vs late-stage (n = 12) groups] after determining the net increase (SF+MSCs–SFNeat) in cytokines due to exposure to a single donor-MSC at the screening stage and to three independent donor-MSCs at the validation stage. Each donor-MSC was tested in duplicate with each SF and dependencies were removed by using the mean of duplicate measurements. Each SF treatment is considered independent, each coming from a different patient. Wilcoxon rank-sum test was used for comparisons between early- and late-stage OA SF groups for each donor-MSC. To account for the overall effect of early- and late-stage OA SFs on all donor-MSCs, we used linear mixed-effects models: with MSC and SF donors modeled as a random factors and SF stage as a fixed factor. P values less than 0.05 were considered to be statistically significant. OA stage effect sizes (r and marginal R2, mR2) were calculated as described in Supplementary Methods. All statistical analyses were performed with R version 3.1.2.
Since little is known on the differential protein secretion of MSCs in response to stage specific OA SFs (with only data on the effect of OA SFs on MSC gene expression
Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.
), we first screened the secretome of a single donor-MSC against a panel of 64 cytokines in response to early- (n = 12) and late-stage (n = 11) OA SFs (Supplementary Table 2). We confirmed that MSCs remain viable and proliferate when cultured in both early- and late-stage OA SFs (Supplementary Fig. 3). From the analytes detected using a 64-plex immunoassay, nine proteins were present in measurable and similar levels within the MSC secretome, after exposure to both early- and late-stage OA SF: CCL2, chemokine (C-C motif) ligand-3 (CCL3), chemokine (C-C motif) ligand-7 (CCL7), chemokine (C-X3-C motif) ligand-1 (CX3CL1), CXCL8, chemokine (C-X-C motif) ligand-1/2/3 (CXCL1/2/3) and chemokine (C-X-C motif) ligand-5 (CXCL5); and the cytokines: vascular endothelial growth factor A (VEGFA) and IL6 (Supplementary Table 3). However only two cytokines, CXCL12 (r = −0.452, P = 0.027) and CXCL8 (r = 0.616, P = 0.002) were differentially secreted by MSCs in the presence of early- vs late-stage OA SF, with CXCL8 being higher and CXCL12 lower in early-stage OA SF (Supplementary Table 3).
Validation
Based on this initial screening, we identified four cytokines to analyze further. IL6 and CCL2 were selected as they are the most abundantly secreted cytokines; CXCL8 and CXCL12 were selected as they were the only two differentially secreted by MSCs in early- vs late-stage OA SF.
Secretome analysis on three donor-MSCs confirmed high levels of IL6 and CCL2 secretion in both OA stages, with a higher overall secretion in early- vs late-stage OA for both IL6 (mR2 = 0.07 and P = 0.018) and CCL2 (mR2 = 0.05 and P = 0.023) [Fig. 2(A) and (B)]. While IL6 was abundantly secreted by MSCs (>3 ng/mL), irrespective of the donor tested, CCL2 secretion was more variable and MSC-donor dependent, with medians from all OA SFs of 1137, 569 and 374 pg/mL for MSC-1, -2 and -3, respectively. CXCL8 secretion by MSCs was confirmed to be at higher levels after exposure to early- vs late-stage (mR2 = 0.142, P < 0.001); analysis of each donor-MSC showed an effect of the OA stage of SFs in two out of three donor-MSCs [Fig. 2(C)]. However, differential secretion of CXCL12 was not confirmed in MSCs from all three donors [Fig. 2(D)], unlike what was seen in the screening phase.
Fig. 2Selected cytokines secreted by MSCs in early- and late-stage OA SFs. Cytokines are shown as fold change from the baseline MSC secretion (dotted line) in DMEM with 0.5% FBS: with IL6 at 1513 [1291, 1843] pg/mL; CCL2 at 1946 [1706, 2084] pg/mL; CXCL8 at 71 [46, 387] pg/mL; and CXCL12 at 199 [197, 279] pg/mL. Individual values are shown with median and ranges indicate interquartile range. P values shown indicate comparison between the variation of cytokine levels in early- (n = 12) vs late-stage OA (n = 12) SF after exposure to MSCs.
Our results indicate for the first time that MSCs have a differential response to early- vs late-stage OA, with a higher secretion of CXCL8, IL6 and CCL2 in early-stage OA SFs. With OA-stage having the biggest effect on CXCL8 followed by IL6 (Supplementary Tables 5 and 6). Identifying the MSC secretome
in the context of different OA microenvironments (early- vs late-stage OA) is an important first step in determining the therapeutic utility of MSCs for OA.
Previous reports show the effect of OA SF on MSCs, both human
Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.
Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.
. However, a systematic analysis of MSC secretome in response to stage-specific OA SF has not been reported. In this study we report that the secretion levels of MSC-derived cytokines are differentially modulated by OA microenvironments. It is important to keep in mind that our results are limited to OA SFs from patients with symptomatic OA that is severe enough to require intervention, which is the population that would likely be a candidate for MSC-based therapy.
Our results indicate an increase in IL6 secretion by MSCs in OA SFs, being more pronounced in early-stage OA SFs. In animal models of OA, mice lacking IL6 develop more severe OA and IL6 addition decreases proteoglycan loss
. Furthermore, CCL2 and IL6, both of which are secreted at higher levels by MSCs after exposure to early-OA SFs (Fig. 2), can direct M2 polarization of monocyte/macrophages
. This suggests a potential anti-inflammatory mechanism in modulating OA that may be more potent in early-OA patients. Interestingly, we have observed that IL6 is present at higher basal levels in late-stage OA SFs (Supplementary Table 4) while CCL2 basal levels do not differ based on OA-stage (P = 0.072).
CXCL12 secretion by MSCs was found to be highly variable (with coefficients of variation of 123.06–909.78% vs 49.37–148.17% for CXCL8, IL6 and CCL2 combined), likely due to combined MSC and SF donor variability. While CXCL12 levels are increased in OA vs normal SF, with concomitant increase in proteoglycan loss and MMP13 activity in human cartilage
, it is not known how MSC secreted CXCL12 could affect OA progression or repair. However, MSC-dependant CXCL12 variability indicates the potential to pre-select MSCs with higher or lower levels of CXCL12, depending on the population that proves to be more beneficial for OA.
. Interestingly, we observed that CXCL8 is secreted at higher levels by MSCs in early-stage OA SF while it is present at higher levels in late-stage OA neat SF (Supplementary Table 4); there is no negative correlation between neat SF levels of CXCL8 and levels of CXCL8 secreted by MSCs while in SFs. This paradoxical role of CXCL8 in possibly enabling a MSC-mediated effect in OA is unclear, and warrants further investigation.
Taken together, our study demonstrates that MSCs react differently to early- and late-stage OA SFs by differentially secreting factors involved in macrophage polarization, chemotaxis and angiogenesis. These differences may be important in MSC-mediated repair, perhaps being more effective in one stage of OA vs another. While further work is needed, including testing how the MSC-mediated secretion into OA SFs functionally affects joint tissues, our findings are a key step in understanding how MSC modulate the OA microenvironment.
Contributions
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published.
Conception and design of the study: Alejandro Gómez-Aristizábal, Anirudh Sharma, Mohit Kapoor, Penney M Gilbert, Sowmya Viswanathan and Rajiv Gandhi.
Data collection and assembly: Alejandro Gómez-Aristizábal, Anirudh Sharma, Mohsen A Bakooshli.
Statistical analysis: Alejandro Gómez-Aristizábal and Anirudh Sharma.
Analysis and interpretation of data: Alejandro Gómez-Aristizábal, Anirudh Sharma, Mohit Kapoor, Penney M Gilbert, Sowmya Viswanathan and Rajiv Gandhi.
Alejandro Gómez-Aristizábal ([email protected]) and Anirudh Sharma ([email protected]) take responsibility for the integrity of the work as a whole.
Competing interests
None of the authors have any potential conflicts of interest to disclose that could be perceived to influence the present work.
Role of the funding source
None.
Acknowledgements
The authors thank the Krembil Foundation for funding this work. Authors would like to thank study participants including patients. Special thanks to Dr Xing Hua Wang and Dr Armand Keating for providing MSCs for this project and Kala Sundararajan for her guidance on statistics.
Appendix A. Supplementary data
The following is the supplementary data related to this article:
Inflammatory stimuli differentially modulate the transcription of paracrine signaling molecules of equine bone marrow multipotent mesenchymal stromal cells.
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