Study of the microRNA expression profile of foreskin derived mesenchymal stromal cells following inflammation priming
- Hussein Fayyad-Kazan†1Email author,
- Mohammad Fayyad-Kazan†2,
- Bassam Badran1,
- Dominique Bron3,
- Laurence Lagneaux3 and
- Mehdi Najar3
© The Author(s) 2017
Received: 28 February 2016
Accepted: 6 December 2016
Published: 13 January 2017
Due to their self-renewal capacity, multi-lineage potential, and immunomodulatory properties, mesenchymal stromal cells (MSCs) are an attractive tool for different therapeutic strategies. Foreskin (FSK), considered as a biological waste material, has already been shown to be a valuable source of MSCs. Besides their typical fibroblast like morphology and International Society for cellular Therapy compliant phenotype, foreskin-MSCs (FSK–MSCs) are clonogenic, and highly proliferative cells with multi-lineage and strong immunomodulatory capacities. Of importance, FSK–MSCs properly adjust their fate following exposure to inflammatory signals. Being potent regulators of gene expression, miRNAs are involved in modulating nearly all cellular processes and in orchestrating the roles of different immune cells. In this study, we characterized the miRNome of FSK–MSCs by determining the expression profile of 380 different miRNAs in inflammation primed vs. control non-primed cells.
TaqMan low density array (TLDA) was performed to identify dysregulated miRNAs after exposing FSK–MSCs to inflammatory signals. Quantitative real-time RT-PCR was carried out to validate the observations. DIANA-miRPath analysis web server was used to identify potential pathways that could be targeted by the dysregulated miRNAs.
Sixteen miRNAs were differentially expressed in inflammation-primed vs. non-primed FSK–MSCs. The expression level of miR-27a, -145, -149, -194, -199a, -221, -328, -345, -423-5p, -485-3p, -485-5p, -615-5p and -758 was downregulated whilst that of miR-155, -363 and -886-3p was upregulated. Target pathway prediction of those differentially expressed miRNAs identified different inflammation linked pathways.
After determining their miRNome, we identified a striking effect of inflammatory signals on the miRNAs’ expression levels in FSK–MSCs. Our results highlight a potential role of miRNAs in modulating the transcription programs of FSK–MSCs in response to inflammatory signals. Further, we propose that specific miRNAs could serve as interesting targets to manipulate some functions of FSK–MSCs, thus ameliorating their therapeutic potential.
KeywordsMSCs Foreskin Inflammation miRNA
Mesenchymal stromal cells (MSCs) are multipotent fibroblast-like cells found in almost all tissues . This multi-lineage potential, along with their capacity of supporting hematopoiesis and modulating immune responses are the main properties of MSCs. The simple and easy isolation procedures together with the great expansion potential, render MSCs as ideal candidates for different cellular therapies . Despite some common characteristics and properties shared by MSCs of different origin , significant differences in their immunobiological features exist [4, 5]. Although it has long been considered as a biological waste material, foreskin (FSK) is, nowadays, considered as an important reservoir of therapeutic cells having potential value for several clinical applications [6, 7]. Recently, we have demonstrated that the foreskin is a new source of MSCs, designated as FSK–MSCs . Being compliant with ISCT criteria, non-immunogenic and showing powerful immunomodulatory capacities , FSK–MSCs are highlighted as a valuable tolerogenic tool for cell-based immunotherapy. As known, MSCs are particularly sensitive to the local inflammatory microenvironment that modulate their functions and responses. Indeed, inflammation-priming leads to significant changes in the immunobiology of FSK–MSCs .
MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules that regulate the transcription or translation of the target gene mRNAs. Thus, they play crucial roles in many different cellular processes including cell differentiation, proliferation and immune homeostasis [9–12]. Accumulating data show that abnormal miRNA expression is a common feature of various diseases [9–12]. Nowadays, many evidences demonstrate a role for miRNAs in regulating distinct aspects of the immune system including cell proliferation, differentiation, fate determination and function mediated by different cell types [13–15]. The understanding of the miRNome of FSK–MSCs under inflammatory settings will help to identify the pattern of miRs that are modulated and that could serve as targets to enhance MSCs therapeutic functions. In this study, comparative analysis showed that the miRNA expression profile of FSK–MSCs is critically different in inflammation primed vs. control non-primed cells. Our results reveal specific miRNA expression differences following inflammation priming and identified 16 differentially expressed miRNAs. Those altered miRNAs might be involved in the molecular mechanisms regulating FSK–MSCs immunobiology during inflammatory conditions and could be further used as targets to manipulate FSK–MSCs functions.
This study was conducted in accordance with the Declaration of Helsinki (1964) and approved by the local ethics committee of the “Institut Jules Bordet” (Belgium). All donors and/or their parents or legal guardian were voluntary for giving the foreskin (FSK) samples as obtained following a circumcision procedure from healthy subjects. They gave written informed consent before specimen collection for using the samples and for publishing any associated scientific data. However, all personal information are kept confidential.
Isolation, culture and characterization of FSK–MSCs
Briefly, after circumcision, foreskin was aseptically collected into a sterile specimen container containing sterile phosphate-buffered saline (PBS) buffer supplemented with penicillin/streptomycin. The samples were processed as previously published . After a sterile wash with PBS, the sample was transferred into a petri dish where epidermis was manually removed from the skin and the dermis was cut into small pieces. By using Liberase Research Grade solution (Roche Diagnostics, Belgium), the tissue was digested and the resulting cell suspension was washed by centrifugation (800g, 5 min) in Dulbecco’s modified Eagle’s medium with low glucose (DMEM-LG; Lonza, Belguim) and containing 10% fetal bovine serum (FBS; Sigma-Aldrich, Belgium). The subsequent cell pellet was seeded in culture flasks with DMEM-LG (Lonza) supplemented with 10% FBS (Sigma-Aldrich), 2 mM l-glutamine and 50 U/ml penicillin (both from Lonza) and the cultures were incubated at 37 °C in a 5% CO2 humidified atmosphere. Non-adherent cells were removed when the medium was changed (once a week). When sub-confluence (80–90%) was achieved, adherent cells were harvested by TrypLE Select (Lonza) and expanded until the desired passage.
The cells were characterized according to the ISCT criteria. Thus, the immunophenotype was analysed by flow cytometry (MacsQuant analyzer (Miltenyi Biotec, Netherlands)) using fluorochrome labelled monoclonal antibodies: anti-CD45-FITC and anti-HLA-Dr-PE (Exalpha Biologicals, Maynard, MA), anti-CD34-PE and anti-CD73-PE (BD Biosciences, San Diego, CA, USA), anti-CD14-PE, anti-CD19-PE, anti-CD105-FITC and anti-CD90-PE (R&D systems, Minneapolis, MN, USA). Their multilineage potential was confirmed by inducing specific lineage commitment (adipogenic, osteogenic and chondrogenic differentiation) using appropriate induction culture medium (NH media, Miltenyi Biotec). Specific lineage commitment was highlighted by using lineage-specific cell staining techniques and microscopic examination.
Inflammation priming of FSK–MSCs
Foreskin–mesenchymal stromal cells were analyzed under both constitutive and inflammatory conditions. Inflammation priming of FSK–MSCs was performed as previously described . Briefly, cells were treated (overnight) with a pro-inflammatory cytokine cocktail containing IL-1β (Peprotech, Rocky Hill, NJ, USA) (25 ng/ml), TNF-α (50 ng/ml), IFN-α (3000 U/ml or 10 ng/ml) and IFN-γ (1000 U/ml or 50 ng/ml) (all from Prospec Inc., Rehovot, Israel). After priming, the medium was removed, and the cells were washed and became available for analysis.
MiRNA expression profile
Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science). The concentration was quantified using a NanoDrop spectrophotometer. A three-step procedure was performed to profile the miRNAs. First, for cDNA synthesis from the miRNAs, 30 ng of total RNA was subjected to RT (reverse transcription) using a TaqMan® microRNA Reverse Transcription Kit (#4366596; Applied Biosystems) and Megaplex RT primers (Human Pool A, #4399966; Applied Biosystems) following the manufacturer’s protocol, allowing simultaneous reverse transcription of 380 mature human miRNAs to generate a miRNA cDNA library corresponding to each plasma sample. RT was performed on a Mastercycler Epgradient thermocycler (Eppendorf) with the following cycling conditions: 40 cycles at 16 °C for 2 min, 42 °C for 1 min and 50 °C for 1 s followed by a final step of 80 °C for 5 min to inactivate reverse transcriptase. Thereafter, to generate enough miRNA cDNA template for the following real-time PCR, the cDNA libraries were pre-amplified using Megaplex PreAmp primer (Humam Pool A, #4399233; Applied Biosystems) and PreAmp Master Mix (#4384266; AppliedBiosystems) following the manufacturer’s instructions. The PreAmp primer pool used here consisted of forward primers specific for each of the 380 human miRNAs and a universal reverse primer. The pre-amplification cycling conditions were as follows: 95 °C for 10 min, 55 °C for 2 min, 72 °C for 2 min followed by 12 cycles at 95 °C for 30 s and 60 °C for 4 min; the samples were then held at 99.9 °C for 10 min. After the pre-amplification step, the products were diluted with RNase-free water, combined with TaqMan gene expression Master Mix and then loaded into TaqMan Human MicroRNA Array A (#4398965; Applied Biosystems), which is a 384-well formatted plate and real-time PCR-based microfluidic card with embedded TaqMan primers and probes in each well for the 380 different mature human miRNAs. Real-time PCR was performed on an ABI PRISM 7900HT sequence detection system (Applied Biosystems) with the following cycling conditions: 50 °C for 2 min, 94.5 °C for 10 min followed by 40 cycles at 95 °C for 30 s and 59.7 °C for 1 min. The Ct (cycle threshold) was automatically given by SDS 2.4 software (Applied Biosystems) and is defined as the fractional cycle number at which the fluorescence passes the fixed threshold of 0.2. RNU48 embedded in the TaqMan human microRNA arrays was used as an endogenous control. The relative expression levels of miRNAs were calculated using the comparative ΔΔCt method as described previously [17, 18]. The fold changes in miRNAs were calculated by the equation 2−ΔΔCt.
Taqman miRNA assay for individual miRNAs
Gene-specific reverse transcription was performed for each miR using 10 ng of purified total RNA, 100 mM dNTPs, 50 U MultiScribe reverse transcriptase, 20 U RNase inhibitor, and 50 nM of gene-specific RT primer samples using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Gent, Belgium). 15 μl reactions were incubated for 30 min at 16 °C, 30 min at 42 °C, and 5 min at 85 °C to inactivate the reverse transcriptase. Real time RT-PCR reactions (5 μl of RT product, 10 μl TaqMan 2 × Universal PCR master Mix, (Applied Biosystems, Gent, Belgium), and 1 μl TaqMan MicroRNA Assay Mix containing PCR primers and TaqMan probes) were carried out on ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Gent, Belgium) at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The qRT-PCR reactions were performed in triplicate, and the signal was collected at the end of every cycle. The Ct (cycle threshold) was automatically given by SDS 2.4 software (Applied Biosystems) and is defined as the fractional cycle number at which the fluorescence passes the fixed threshold of 0.2.
RNU48 was used as an endogenous control. The relative expression levels of miRNAs were calculated using the comparative ΔΔCt method as described previously [31, 32]. The fold changes in miRNAs were calculated by the equation 2−ΔΔCt.
Pathway enrichment analysis
Pathway enrichment analysis was employed to investigate the regulatory mechanisms of significantly differentially expressed miRNAs. DIANA-miRPath v.3.0 analysis web server was used for pathway enrichment analyses. The enriched pathways were defined by their enrichment of significantly differentially expressed miRNA target genes.
Data analyses were performed using the SDS RQ Manager 1.2 software and DataAssist v2.0 software (Applied Biosystems). Statistical significance of miRNA expression between control and treated cells was determined according to unpaired Mann–Whitney U test. p Values <0.05 (*), <0.01 (**) were considered significant.
Obtaining and characterizing FSK–MSCs
In order to obtain FSK–MSCs, the foreskin sample was first processed to separate the epidermis from the dermis (see "Methods" section) and then scraping was applied to obtain small tissue pieces from the dermis. Following enzymatic digestion, the tissue mixture was centrifuged and the resulting cell pellet was incubated for culture. After removing non adherent cells, colonies of cells with fibroblastic morphology started to appear. When sub-confluency (80–90%) was reached, adherent cells were harvested using TrypLE Select solution and expanded by successive passages.
These adherent cells were then characterized according to the ISCT criteria. Flow cytometry analysis demonstrated that those cells are positive (>95%) for the MSC markers CD73, CD90 and CD105 whilst negative (<5%) for CD45, CD34, CD14, CD19 and HLA-DR markers. It is noteworthy that inflammation priming had no significant impact on the expression level of those markers (data not shown).
To comply with the ISCT criteria, we further confirmed that the obtained cells exhibited a multi-lineage potential since they were able to differentiate into cells of adipogenic, osteogenic or chondrogenic lineage after being cultured with appropriate induction media (data not shown). After 21 days of osteogenic differentiation, a mineralization matrix following calcium deposits was observed by Alizarin red staining demonstrating the generation of osteoblasts. After 10 days of adipogenic differentiation, cells with cytoplasm containing several lipid vacuoles were revealed by Oil Red O staining indicating the formation of adipocytes. After 21 days of chondrogenic differentiation, the production of proteoglycans-based extracellular matrix was shown by Alcian blue staining illustrating the generation of chondrocytes.
Impact of inflammation priming on miRNA-expression profile in FSK–MSCs cells
MiRNA signature identified by TLDA Technique
Inflammation vs. Ctrl ratio
The numbers corresponding to these colors are the ΔCt values. The dendrogram on the left side of the heat map classifies miRNAs into groups based on the divergence of miRNA expression values among the different samples. The dendrogram presented at the top indicates the relatedness of the samples based on overall miRNA expression values and separates the control from the treated group of samples.
On the other hand, group 2 contains 3 miRNAs (miR-155, -363 and -886-3p) that were upregulated (ratio greater than 3) in treated vs. control cells (Fig. 2). Among these, miR-155 was the most strikingly upregulated miR exhibiting a 9.4 fold increase whilst miR-363 and -886-3p showed increased rates of 4.7 and 4.5 folds, respectively. Altogether, these observations demonstrate a clear difference in the miRNA expression profile in FSK–MSCs exposed to inflammatory signals vs. control cells suggesting a potential role for miRNAs in modulating FSK–MSCs’ transcriptional programs in response to inflammatory conditions.
Analysis of inflammation primed MSCs–associated miRNA pathways
Since each miRNA can regulate the expression of many target genes but multiple miRNAs can modulate specific pathways, we explored the pathways that were potentially regulated by the miRNAs observed to be altered in inflammation primed FSK–MSCs in comparison to untreated control MSCs.
Significantly enriched KEGG pathways (p < 0.05) targeted by miRNAs
PI3-AKT signaling pathway
Apoptosis signaling pathway
Wnt/Ca2+ signaling pathway
6.9 x 10−8
PI3-AKT signaling pathway
Apoptosis signaling pathway
Apoptosis signaling pathway
Leukocyte transendothelial migration
Wnt/Ca2+ signaling pathway
NF-Kappa B signaling
TNF signaling pathway
In addition to their self-renewing and multi-lineage differentiation properties, MSCs exhibit immunoregulatory abilities through influencing both immune and inflammatory responses . In fact, MSCs, can actively sense the surrounding microenvironment or inflammatory milieu and adopt their phenotype and responses, accordingly . For instance, and depending on the environmental signals, MSCs can play both pro- and anti-inflammatory roles . This marked functional plasticity of MSCs renders them as clinically relevant cell types with important therapeutic potential for tissue repair and regeneration as well as for the treatment of different inflammatory diseases and malignancies [20, 21]. Nowadays, different populations of MSCs, derived from distinct human tissues, are available and exhibit dissimilar immunological profiles and functional capacities [5, 22, 23]. FSK–MSCs are emerging as a highly interesting MSC-type due to their recently characterized properties . Besides, FSK–MSCs, when exposed to inflammatory conditions, have been shown to exhibit induced expression of different immunoregulatory genes and factors . Their high sensitivity to inflammation increased the interest in understanding the molecular mechanisms involved in coupling FSK–MSCs’ responses to the surrounding inflammatory microenvironment.
Among the different regulatory elements that controls gene expression and modulates cellular fate and behavior are miRNAs. For instance, they can significantly alter the responses of different innate and adaptive immune cells where any disturbance of their expression profiles can lead to several diseases [24–27]. Therefore, miRNAs are, nowadays, considered as attractive therapeutic targets [24–27]. Besides their ability to modulate immune responses, miRNAs play a key role in MSCs’ biology. For example, miRNAs can strikingly affect MSCs’ decision to either proliferate or differentiate into either cell lineage including adipogenic, chondrogenic, myogenic, neurogenic or other lineages . The aim of this work was to determine the miRNome of FSK–MSCs by investigating the expression profile of 380 different miRNAs in inflammation primed vs. control non-primed cells. The major outcome of this study is our demonstration of a significantly altered miRNA expression profile in inflammation-primed FSK–MSCs where 16 miRNAs were found to be differentially expressed. This observed inflammation-triggered alteration of miRNA expression pattern is not surprising especially that different miRNAs have been reported to show altered expression in response to inflammatory signals. For example, miR-146 expression level is described to be upregulated in response to inflammatory stimuli , whilst other miRNAs including miR-9, -101, -125b, -155, -146, -192 and -203 are described to exhibit altered expression profiles in some inflammatory diseases .
We classified our differentially expressed miRNAs in two major groups: (1) a first one containing the miRNAs being downregulated in inflammation-primed FSK–MSCs and includes miR-27a, -145, -149, -194, -199a, -221, -328, -345, -423-5p, -485-3p, -485-5p, -615-5p and -758; (2) and a second one bearing the upregulated miRNAs and consists of miR-155, -363 and -886-3p. Interestingly, among those identified miRNAs, there are ones that have already been demonstrated to regulate MSCs’ behavior. For instance, miR-27a has been reported to regulate osteogenesis of MSCs  whilst miR-145, -194, and -199a have been described to regulate their chondrogenesis [32–34]. Moreover, miR-221 has been shown to be involved in the adipogenesis process of MSCs . Besides its ability to regulate adipogenesis , miR-155 has also been described to modulate the responses of MSCs to inflammatory signals . Furthermore, miR-886-3p overexpression has been shown to inhibit MSCs migration .
Given that a variety of genes, encoding cell adhesion molecules, co-stimulatory elements as well as immunoregulatory cytokines and factors, are modulated in inflammation-primed FSK–MSCs, it is strongly possible that the differentially expressed miRNAs could be involved in the regulatory processes accounting for such altered transcription programs in inflammation-primed vs. non-primed FSK–MSCs. This proposal is strongly supported by the observation that miR-155 expression can be modulated upon inflammation-priming of mouse MSCs and that miR-155 expression levels are tightly related to its ability to modulate the immunosuppressive capacity of those MSCs , thus providing a striking evidence for the importance of miRNAs in regulating the immunoregulatory capacity of MSCs. To get more insight into the potential mechanisms and signaling events that could be targeted by our differentially expressed miRNAs, we searched for the predicted target pathways of those miRNAs. In fact, given that multiple miRNAs may cooperate to regulate related target genes in a certain signaling route [38, 39], analysis of target pathways instead of individual genes was carried out. The miRNAs, being downregulated in inflammation-primed FSK–MSCs, are predicted to target pathways enriched in genes involved in Adherens junction, PI3-AKT pathway, apoptosis pathway, Wnt/Ca2+ pathway and leukocyte trans-endothelial migration. On the other hand, adherens junction, TNF and NF-KB signaling pathways are predicted to be the targets of miR-155 which is upregulated in inflammation-primed FSK–MSCs. Indeed, it was previously reported that miR-155, being upregulated in inflammation primed mouse MSCs, modulates MSCs immunosuppressive capacity upon modulating NF-KB activity .
The observed altered miRNA expression profile in inflammation-primed vs. non-primed FSK–MSCs imposes a detailed mechanistic characterization of the involvement of each of the identified miRNAs as well as their predicted pathway(s) on FSK–MSCs inflammatory responses in future studies. However, our observations might provide an early step toward developing a novel strategy to improve the immunosuppressive activity of FSK–MSCs. For instance, identifying the miRNAs that either dampen or trigger the expression of genes encoding immunosuppressive cytokines, factors and receptors would enable us to trigger, in vitro, the immunomodulatory capacity of FSK–MSCs by downregulating or upregulating the expression of certain miRNAs. This, in vitro, pre-activation of FSK–MSCs would greatly improve their therapeutic potential and medical value.
In this report, we show that inflammation-priming of FSK–MSCs substantially alters their miRNA expression profile where 13 miRNAs are downregulated and 3 others are upregulated. Those differentially expressed miRNAs are predicted to target several candidate signaling pathways being involved in regulating cellular behavior in response to inflammatory signals.
Aware that the in vivo communication between grafted MSCs and the host inflammatory microenvironment orchestrates the plasticity of the immunological properties of the grafted MSCs, understanding the molecular events that transplanted MSCs undergo upon exposure to an inflammatory signal is of great importance. In this report, we highlight a potential role for miRNAs in regulating FSK–MSCs’ responses to inflammatory signals. Further, we suggest that miRNAs could serve as potential targets to improve, in vitro, the therapeutic value of MSCs before being grafted into the patient.
G protein-coupled receptor
International Society for cellular Therapy
Kyoto encyclopedia of genes and genomes
- MSCs :
mesenchymal stromal cells
peroxisome proliferator-activated receptor
- qRT-PCR :
quantitative Real-Time PCR
tumor necrosis factor
TaqMan low density array
HFK and BB conceived and designed the study, performed experiments, acquired data, analyzed the data, interpreted data, drafted the manuscript and revised the manuscript. MFK collected and provided samples, performed experiments, acquired data, analyzed the data, interpreted data, drafted the manuscript and revised the manuscript. DB, LL and MN conceived and designed the study, collected and provided samples, interpreted data, drafted the manuscript and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and supporting materials
Authors do not wish to share their data as they are confidential.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Clark EA, Kalomoiris S, Nolta JA, Fierro FA. Concise review: MicroRNA function in multipotent mesenchymal stromal cells. Stem Cells. 2014;32:1074–82.View ArticlePubMedGoogle Scholar
- da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev. 2009;20:419–27.View ArticleGoogle Scholar
- Via AG, Frizziero A, Oliva F. Biological properties of mesenchymal stem cells from different sources. Muscles Ligaments Tendons J. 2012;2:154–62.PubMedGoogle Scholar
- De Kock J, Najar M, Bolleyn J, Al Battah F, Rodrigues RM, Buyl K, et al. Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential. Stem Cells Dev. 2012;21:3309–23.View ArticlePubMedGoogle Scholar
- Najar M, Raicevic G, Fayyad-Kazan H, Kazan HF, De Bruyn C, Bron D, et al. Immune-related antigens, surface molecules and regulatory factors in human-derived mesenchymal stromal cells: the expression and impact of inflammatory priming. Stem Cell Rev. 2012;8:1188–98.View ArticlePubMedGoogle Scholar
- Ashley F. Foreskins as skin grafts. Ann Surg. 1937;106:252–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Zaroo MI, Sheikh BA, Wani AH, Darzi MA, Mir M, Dar H, et al. Use of preputial skin for coverage of post-burn contractures of fingers in children. Indian J Plast Surg. 2011;44:68–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Najar M, Raicevic G, André T, Fayyad-Kazan H, Pieters K, Bron D, et al. Mesenchymal stromal cells from the foreskin: tissue isolation, cell characterization and immunobiological properties. Cytotherapy. 2016;18:320–35.View ArticlePubMedGoogle Scholar
- Pallante P, Visone R, Croce CM, Fusco A. Deregulation of microRNA expression in follicular-cell-derived human thyroid carcinomas. Endocr Relat Cancer. 2010;17:F91–104.View ArticlePubMedGoogle Scholar
- Schetter AJ, Heegaard NHH, Harris CC. Inflammation and cancer: interweaving microRNA, free radical, cytokine and p53 pathways. Carcinogenesis. 2010;31:37–49.View ArticlePubMedGoogle Scholar
- Miller BH, Wahlestedt C. MicroRNA dysregulation in psychiatric disease. Brain Res. 2010;1338:89–99.View ArticlePubMedPubMed CentralGoogle Scholar
- Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, et al. miRNA malfunction causes spinal motor neuron disease. Proc Natl Acad Sci US A. 2010;107:13111–6.View ArticleGoogle Scholar
- Lindsay MA. microRNAs and the immune response. Trends Immunol. 2008;29:343–51.View ArticlePubMedGoogle Scholar
- Chan CWY, Kay LS, Khadaroo RG, Chan MWC, Lakatoo S, Young KJ, et al. Soluble fibrinogen-like protein 2/fibroleukin exhibits immunosuppressive properties: suppressing T cell proliferation and inhibiting maturation of bone marrow-derived dendritic cells. J Immunol. 2003;170:4036–44.View ArticlePubMedGoogle Scholar
- Lodish HF, Zhou B, Liu G, Chen C-Z. Micromanagement of the immune system by microRNAs. Nat Rev Immunol. 2008;8:120–30.View ArticlePubMedGoogle Scholar
- Raicevic G, Rouas R, Najar M, Stordeur P, Boufker HI, Bron D, et al. Inflammation modifies the pattern and the function of toll-like receptors expressed by human mesenchymal stromal cells. Hum Immunol. 2010;71:235–44.View ArticlePubMedGoogle Scholar
- Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc. 2008;3:1101–8.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-delta delta C(T)) method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Loza MJ, McCall CE, Li L, Isaacs WB, Xu J, Chang B-L. Assembly of inflammation-related genes for pathway-focused genetic analysis. PLoS ONE. 2007;2:e1035.View ArticlePubMedPubMed CentralGoogle Scholar
- Bernardo ME, Fibbe WE. Mesenchymal stromal cells: sensors and switchers of inflammation. Cell Stem Cell. 2013;13:392–402.View ArticlePubMedGoogle Scholar
- Yagi H, Kitagawa Y. The role of mesenchymal stem cells in cancer development. Front Genet. 2013;4:261.View ArticlePubMedPubMed CentralGoogle Scholar
- Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood. 2001;98:2396–402.View ArticlePubMedGoogle Scholar
- El Omar R, Beroud J, Stoltz J-F, Menu P, Velot E, Decot V. Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies? Tissue Eng Part B Rev. 2014;20:523–44.View ArticlePubMedGoogle Scholar
- Jeker LT, Bluestone JA. MicroRNA regulation of T-cell differentiation and function. Immunol Rev. 2013;253:65–81.View ArticlePubMedPubMed CentralGoogle Scholar
- He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S, et al. The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci US A. 2005;102:19075–80.View ArticleGoogle Scholar
- Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, et al. Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol. 2008;9:405–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Raisch J, Darfeuille-Michaud A, Nguyen HTT. Role of microRNAs in the immune system, inflammation and cancer. World J Gastroenterol. 2013;19:2985–96.View ArticlePubMedPubMed CentralGoogle Scholar
- Collino F, Bruno S, Deregibus MC, Tetta C, Camussi G. MicroRNAs and mesenchymal stem cells. Vitam Horm. 2011;87:291–320.View ArticlePubMedGoogle Scholar
- Taganov KD, Boldin MP, Chang K-J, Baltimore D. NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA. 2006;103:12481–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Sonkoly E, Pivarcsi A. Advances in microRNAs: implications for immunity and inflammatory diseases. J Cell Mol Med. 2009;13:24–38.View ArticlePubMedGoogle Scholar
- Hassan MQ, Gordon JAR, Beloti MM, Croce CM, van Wijnen AJ, Stein JL, et al. A network connecting Runx2, SATB2, and the miR-23a ~ 27a ~ 24-2 cluster regulates the osteoblast differentiation program. Proc Natl Acad Sci USA. 2010;107:19879–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Martinez-Sanchez A, Dudek KA, Murphy CL. Regulation of human chondrocyte function through direct inhibition of cartilage master regulator SOX9 by microRNA-145 (miRNA-145). J Biol Chem. 2012;287:916–24.View ArticlePubMedGoogle Scholar
- Xu J, Kang Y, Liao W, Yu L. MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS ONE. 2012;7:e31861.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin EA, Kong L, Bai X-H, Luan Y, Liu C-J. miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smad1. J Biol Chem. 2009;284:11326–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Skårn M, Namløs HM, Noordhuis P, Wang M-Y, Meza-Zepeda LA, Myklebost O. Adipocyte differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155, microRNA-221, and microRNA-222. Stem Cells Dev. 2012;21:873–83.View ArticlePubMedGoogle Scholar
- Xu C, Ren G, Cao G, Chen Q, Shou P, Zheng C, et al. miR-155 regulates immune modulatory properties of mesenchymal stem cells by targeting TAK1-binding protein 2. J Biol Chem. 2013;288:11074–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Pillai MM, Yang X, Balakrishnan I, Bemis L, Torok-Storb B. MiR-886-3p down regulates CXCL12 (SDF1) expression in human marrow stromal cells. PLoS ONE. 2010;5:e14304.View ArticlePubMedPubMed CentralGoogle Scholar
- Cloonan N, Brown MK, Steptoe AL, Wani S, Chan WL, Forrest ARR, et al. The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition. Genome Biol. 2008;9:R127.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Z, Sall A, Yang D. MicroRNA: an emerging therapeutic target and intervention tool. Int J Mol Sci. 2008;9:978–99.View ArticlePubMedPubMed CentralGoogle Scholar