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ORIGINAL ARTICLE
Year : 2020  |  Volume : 69  |  Issue : 1  |  Page : 37-47

Gene alterations after human adipose-derived stem cell-derived exosome injection in monosodium iodoacetate-induced osteoarthritis rats by microarray analysis


1 Department of Plastic and Reconstructive Surgery, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam; Department of Obstetrics and Gynecology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Cheongju, Korea
2 Department of Biology, School of Life Sciences, Chungbuk National University, Cheongju, Korea

Date of Submission03-Jun-2019
Date of Acceptance16-Jan-2020
Date of Web Publication11-Apr-2020

Correspondence Address:
Dr. Jae Chul Lee
Department of Plastic and Reconstructive Surgery, Seoul National University Bundang Hospital, 82, Gumi-ro 173 Beon-Gil, Bundang-Gu, Seongnam-si, Gyeonggi-do 463707
Korea
Dr. Soo Young Choe
Department of Biology, Chungbuk National University, 52 Naesudong-ro, Heungdeok-gu, Cheongju 361-804
Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JASI.JASI_67_19

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  Abstract 


Introduction: Exosome (exo) is a small extracellular vesicles containing cell-derived factors, and serve to mediate the paracrine effect of cells. Despite the therapeutic potential of human adipose-derived stem cells (hADSCs)-derived exo-related treatment modalities, the molecular parameters needed to define the “paracrine effect” remain largely unknown. Material and Methods: Using high-density oligonucleotide RNA sequencing, the differential gene expression profiles between a fraction of exo (derived from hADSCs) and its mesenchymal stem cells subpopulation were obtained. Results: Of particular interest was a subset of 66 genes preferentially expressed at fivefold or higher in the group treated with exo derived from hADSCs. This subset contained numerous genes involved in the angiogenesis, inflammatory response, immune response, cell cycle, cell death, cell differentiation, cell proliferation, DNA repair, RNA splicing, and secretion functions. In addition, six protein networks were constructed. The interaction network consisted of 57 proteins encoded by upregulated genes. However, the interaction network also consisted of 69 proteins encoded by downregulated genes. Discussion and Conclusion: Our results provide a basis for more reproducible and reliable quality control using genotypic analysis for the definition of hADSCs-exo. Therefore, these results will serve to provide a basis for studies of the molecular mechanisms which control the core properties of hADSCs-exo. Understanding the processes driving hADSCs-exo recruitment could prove invaluable in the development of novel, targeted therapies to selectively inhibit pathological responses in OA.

Keywords: Adipose-derived stem cell, exosome, microarray, monosodium iodoacetate, osteoarthritis


How to cite this article:
Lee JC, Choe SY. Gene alterations after human adipose-derived stem cell-derived exosome injection in monosodium iodoacetate-induced osteoarthritis rats by microarray analysis. J Anat Soc India 2020;69:37-47

How to cite this URL:
Lee JC, Choe SY. Gene alterations after human adipose-derived stem cell-derived exosome injection in monosodium iodoacetate-induced osteoarthritis rats by microarray analysis. J Anat Soc India [serial online] 2020 [cited 2020 May 28];69:37-47. Available from: http://www.jasi.org.in/text.asp?2020/69/1/37/282304




  Introduction Top


Osteoarthritis (OA) is the most common chronic joint disease diagnosed in the general population, a disease characterized by structural and functional alterations of the articular cartilage.[1] Although OA afflicts a large proportion of the population, there exist relatively few effective therapies which serve to alter or inhibit the actual patho-biologic course of the disease.[2] For many years, scientists have been searching for ways to inhibit the destructive process of the disease, or at least to slow the progressive and irreversible joint damage.[3] This is the driving force behind the numerous current and ongoing efforts to develop new tissue engineering-based strategies for the treatment of OA.[4] Exosomes (exos) are small extracellular membrane vesicles (30–100 nm in diameter) with a mechanism of cell-to-cell communication. Exos are the most extensive class of secreted membrane vesicles that carry proteins and RNAs for intercellular communication.[5],[6],[7] Due to their significant and dynamic role in the biology of a living organism, exo has been widely studied as both a biomarker and therapeutic agent.[8],[9] Although exos derived from stem cells have therapeutic potential with regard to the curative treatment of many illnesses, the precise mechanism by which adipose-derived stem cell (ADSC)-derived exo (ADSCs-exo) affect the natural OA progression has not been widely or fully investigated. Gene expression profiling using microarray analysis has become a promising approach for identifying the changes underlying the initiation and progression of complex diseases such as OA, as well as for the discovery of “biomarkers” for diagnostic purposes, disease activity, and response to treatment.[10] Therefore, in this study, we investigated the changes in gene expression and performed construction of the protein regulatory network by microarray analysis after the injection of human adipose-derived stem cells (hADSCs)-exo into monosodium iodoacetate (MIA)-induced OA rats.


  Material and Methods Top


Animals and treatment

Six-week-old male Sprague-Dawley rats, weighing approximately 180–220 g each, were used for this study. All animal experiments conducted in accordance with the National Institutes of Health Guide for the Care Use of Laboratory Animals (NIH publication No. 86-23, revised in 1996). Knee tissue samples were immediately excised and submitted for microarray analysis.

Monosodium iodoacetate-induced osteoarthritis rat model

Under anesthesia, 50 μL of MIA, dissolved in phosphate-buffered saline (PBS) to yield a concentration of 10 mg/mL, was intra-articularly injected through the infrapatellar ligament of the right knee using a 26G needle. Exactly 1 week after MIA injection, rats in group #5 were administered an intra-articular injection of 200 μL human ADSCs-exo. Rats treated with PBS but not with MIA were used as the control group [Table 1].
Table 1: Differentially expressed genes between four groups

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Human adipose-derived stem cells culture

Subcutaneous adipose samples were obtained from normal humans who provided written informed consent to participate in the experiment. Adipose samples obtained from the patients were maintained in PBS containing antibiotics/antimycotics (Invitrogen, USA) and transported to our laboratory within a day. The ADSCs used for the generation of ADSCs-exo were at passage3–4.

Isolation and identification of exosome derived from adipose-derived stem cells-conditioned medium (CM)

To deplete bovine exos from the medium, the culture medium was centrifuged at 100,000 g for 15 h at 4°C and supernatant was used for ADSC culture.[11] Exo isolation, using Exo-Quick exo precipitation kit (System Biosciences, USA), was performed according to the manufacturer's specifications. The collected exosome morphologies were observed by 100 kv transmission electron microscopy (TEM) [Figure 1]a and [Figure 1]b.
Figure 1: Characterization of adipose-derived stem cell-Exosome. (a) Morphologic analysis of adipose-derived stem cells-exo by transmission electron microscopy (in vivo, arrows). Scale bar = 200 nm. (b) Morphologic analysis of adipose-derived stem cells-exo by transmission electron microscopy (in vitro). Scale bar = 40 nm. (c) Detection of CD81 expression in exosome by Exo ELISA

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Quantification of exosome by CD81 ELISA

The concentration of exosome was determined by the amount of total immunoreactive exosome-associated CD81 (ExoELISA™, System Biosciences, Mountain View, CA). Briefly, 50 μl of exosome was immobilized in 96-well microtiter plates and incubated overnight at 37°C (binding step). Plates were washed three times for 5 min using a wash buffer solution and then incubated with primary antibody (CD81) at room temperature (RT) for 1 hr under agitation. Plates were washed and incubated with secondary antibody (1:5000) at RT 1 hr under agitation. Plates were washed and incubated with super-sensitive TMB ELISA substrate at RT for 45 min under agitation. The reaction was terminated using Stop Buffer solution. Absorbance was measured at 450 nm. The number of exosome particles/ml was obtained using an exosomal CD81 standard curve calibrated against number of exosome particles [Figure 1]c.

Injection of adipose-derived stem cells-exo

ADSCs-exos were isolated from the adipose stem cells-culture medium 1 week after MIA injection, and cultured for 4–5 days, as described above. At 1 week post-MIA injection, ADSCs-exo (200 mg/ml) in 200 μL PBS were intra-articularly injected into the right knee using a 26G needle.

RNA extraction and cDNA synthesis

Total RNA was extracted from the right knee samples, which had been stored for 24 h at room temperature, and then refrigerated (−20°C) using a PAXgene knee RNA extraction kit according to the manufacturer's instructions.

Microarray analysis

Each total RNA sample (100 ng) was labeled and amplified using Low Input Quick Amp Labeling Kit (Agilent Technologies, CA, USA). The Cy3-labeled aRNAs were resuspended in 50 μl of hybridization solution (Agilent Technologies, CA, USA).

Statistical analysis

The arrays were analyzed using an Agilent scanner with associated software. Gene expression levels were calculated with Feature Extraction version 10.7.3.1 (Agilent Technologies, Santa Clara, CA, USA). Fold change filters included the requirement that the genes are present in at least 300% of controls for upregulated genes and lower than 300% of controls for downregulated genes.


  Results Top


Comparison of microarray analysis between the monosodium iodoacetate group and the control group

The expression levels of 2729 genes in the MIA treatment group were significantly different from those in the control group. In addition, no significant difference between the positive control and negative control (data not shown) genes was appreciated. The MIA treatment induced a 1.5-fold increase in the expression of 1340 genes (a two-fold increase in the expression of 489 genes) and a 1.5-fold decrease in the expression of 1389 genes (a two-fold decrease in the expression of 502 genes) compared to the control group [Table 1]. Among the upregulated genes [Table 2], two genes were related to angiogenesis, four genes to cell cycle, six genes to cell death, thirteen genes to cell differentiation, four genes to cell proliferation, two genes to RNA splicing, four genes to secretion, and five genes were related to the extracellular matrix. Seven genes were found to be related to the immune response, and five genes were found to be related to the inflammatory response. Twenty-nine genes were down-regulated in the MIA group compared with the control group [Table 3].
Table 2: Upregulated genes expressed by over 500% between monosodium iodoacetate group and control group

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Table 3: Down-regulated genes expressed by over 600% between monosodium iodoacetate group and the control group

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Comparison of microarray analysis of monosodium iodoacetate and adipose-derived stem cells treatment in monosodium iodoacetate-induced osteoarthritis rats

In microarray analysis, 500 genes showed more than two-fold upregulation of expression, while 441 genes were downregulated in the MIA treatment group compared with the ADSCs treatment group [Table 1]. The expressed genes which showed a six-fold increase are summarized in [Table 4]. One gene was related to angiogenesis, five genes to cell death, nineteen genes to cell differentiation, two genes to cell migration, ten genes to the immune response, and three genes were related to the inflammatory response. The genes which showed decreased expression levels are summarized in [Table 5].
Table 4: Up-regulated genes expressed by over 600% between monosodium iodoacetate group and adipose-derived stem cell group

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Table 5: Down-regulated genes expressed by over 600% between monosodium iodoacetate group and adipose-derived stem cell group

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Comparison of microarray analysis of monosodium iodoacetate and adipose-derived stem cells-exo treatment in monosodium iodoacetate-induced osteoarthritis rats

In RNA-sequencing (Seq) analysis, 1017 genes showed more than two-fold upregulation of expression, while 664 genes were downregulated in the MIA treatment group compared with the ADSCs-exo treatment group [Table 1]. The expressed genes which showed a five-fold increase are summarized in [Table 6]. Two genes were related to angiogenesis, four genes to cell cycle, six genes to cell death, thirty-one genes to cell differentiation, four genes to cell proliferation, one gene to DNA repair, six genes to immune response, one gene to the inflammatory response, two genes to RNA splicing, and seven genes were found to be related to secretion. The genes which showed decreased expression levels are summarized in [Table 7].
Table 6: Upregulated genes expressed by over 500% between monosodium iodoacetate group and adipose-derived stem cell-exo group

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Table 7: Down-regulated genes expressed by over 600% between monosodium iodoacetate group and adipose-derived stem cell-exo group

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Total gene heat map

The expression prolifes from the MIA group, ADSCs group and ADSCs-exo group were compared with those of the reference C group. Gene heat maps were produced using the GenePattern genomic analysis platform. Red color indicates overexpression, while the green color indicates underexpression. Through microarray analysis, changes in the expression of genes associated with the cell death, cell differentiation, cell cycle, cell proliferation, inflammatory response, and immune response, were observed [Figure 2].[12]
Figure 2: Gene heat map in monosodium iodoacetate-induced osteoarthritis rats and human adipose-derived stem cells-exo injection. The expression profiles of heat shock-activated genes were clustered hierarchically and are displayed using a red-green heat map. Heat map of genes upregulated and down-regulated ≥2 fold in monosodium iodoacetate-induced osteoarthritis rats and human adipose-derived stem cells-exo injection. Red color indicates over-expression, while the green color indicates under-expression. Control, control group; monosodium iodoacetate, monosodium iodoacetate group; adipose-derived stem cell, human AD-mesenchymal stem cells group; adipose-derived stem cells-exo, human adipose-derived stem cells-derived exosome group

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The construction of protein regulation network

The protein regulatory network is presented in [Figure 3]. The results revealed that there were several protein genes with differential expression in the control group compared to the MIA group [Figure 3]a and [Figure 3]b. The change in network graph of several proteins was identified after the injection of human ADSCs [Figure 3]c and [Figure 3]d and human ADSCs-exo [Figure 3]e and [Figure 3]f. Furthermore, six protein networks were constructed [Figure 3]. The protein network consisted of 57 red nodes and 69 green nodes. The results revealed that the interactive relationships among proteins are relatively simple.
Figure 3: Protein-protein interaction network. (a-f) Protein network graphs of the biological processes based on Tables 2-7. Red nodes indicate upregulated proteins (a, c, e). Green nodes represent down-regulated proteins (b, d, f). (a) Faslg, Stat6, Prkce, Cybb, Ccl17, Edn1, Ccl22, Cx3cr1, Cxcr2, Ccl25, Ccl6, F2rl1, and Ctsg proteins. (b) Klrk1, Cd27, Klrd1, RT1-CE1, RT1-CE10, Cd69, Gzmb, RT1-A2, Cd8b, Kit, Cd8a, Eif2ak2, Il2ra, Prf1, Cd4, Oas1a, Isg15, Eif2ak2, Il5, Il33, Oas1, Irf7, Stat1, Il23a, Mx2, Oas1k, Mx1, Gbp2, Ccl12, Ccl5, Cxcl10, Tnf, Xcl1, Ccl4, Cxcl9, Cxcl1, Ccl7, Cxcl11, Ccl20, Cxcl13, Ccl27, and Ppbp proteins. (c) Ccl21, Ccl19, Ccl27, Ccl20, Cxcl11, Cxcl13, Cxcl9, Cxcl5, Ccl12, Ccl7, Gbp2, Cxcl10, Ccl11, Isg15, Mx1, Oasl, Oas1a, Mx2, Oas1k, Il5, Il33, Il2ra, Cd40lg, and Gzmb proteins (d) Cxxl2, Cxcr2, Ccr10, Ccr6, Jak3, Il12rb1, Prkce, Igf1r, RT1-T24-4, RT1-CE10, RT1-CE4, and RT1-CE1 proteins (e) Ccl27, Ccl21, Cxcl9, Vegfa, Tnfsf4, Ccl11, Edn1, Kit, RT1-Dob, RT1-Bb, Cd86, Il12b, Gzmb, Gata3, Bcl6, Il5, Il20rb, Ctf1, Il33, and Hras proteins. (f) Cd69, Il7, Ccl9, Cxcl13, Cx3cr1, Ccr6, Ccr10, Yes1, Jak2, Stat2, Faslg, Prkce, Il18, Tnfrsf11b, Il12rb1, and Il12a proteins. Control, control group; monosodium iodoacetate, monosodium iodoacetate group; adipose-derived stem cell, human AD-mesenchymal stem cells group; adipose-derived stem cells-exo, human adipose-derived stem cells-derived exosome group

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  Discussion Top


In this study, it was demonstrated that hADSCs-exo, which were introduced through injection into the intra-articular space and through the infrapatellar ligament, were engrafted in the right knee tissues of MIA-induced OA rats. Adverse effects were not observed after the transfusion of hADSCs (2 × 106/mL/cm2) and hADSCs-exo [Figure 1]. Recently, many papers reported that exos isolated from mouse bone marrow-derived mesenchymal stem cells (MSCs) and exo secreted by induced pluripotent stem cell-derived MSCs (iMSC-Exos) may represent a novel therapeutic effect on OA animal model.[13],[14] However, most of these studies were limited to macroscopic and histological observations of the articular cartilage after stem cell-derived exo treatment. No information was obtained or provided with respect to the effects of these exos on the immune response and inflammatory processes in the synovial membrane and menisci, which occur secondary to OA. This study, using a rat model of OA, was designed to investigate the role of hADSCs-exo in the context of OA and their effect or influence on the inflammatory environment within the affected joint articulation. A more recent publication described that exo from embryonic stem cell-derived MSCs impeded cartilage destruction in the Destabilization of Medial Meniscus model.[15] This concept is further corroborated by a recent study findings said to demonstrate evidence that IL1 β-pretreated MSCs could induce macrophage polarization toward an M2 phenotype more efficiently than naive MSCs, and that miR-146a-containing exo enhanced this effect.[16] In addition, the difference in gene expression levels after human ADSCs-exo therapy was compared in an effort to identify potential candidate genes which might potentially link the systemic immune response to the development of OA disease by examining the gene expression patterns between the MIA group and the ADSCs-exo group in an MIA-induced OA rat model. In the present study, many immunologic processes and genetic factors were thought to be implicated in the pathogenesis of OA. Immunologic abnormalities in the MIA group of OA rats reflected marked activation of the immune system, leading to increased cytokine production. The data indicated that there were several genes with differential expression in the ADSCs-exo group when the ADSCs-exo group was compared to the MIA group. The change in expression levels of several genes was confirmed after the injection of hADSCs-exo. These genes were related to the angiogenesis, inflammatory response, immune response, cell cycle, cell death, cell differentiation, cell proliferation, DNA repair, RNA splicing, and secretion. Despite the injection of hADSCs-exo into MIA-induced OA rats, relatively little is known about the relationship between OA and ADSCs-exo. Therefore, in the current study, it was our intention to more fully investigate the changes in gene expression by microarray analysis after the injection of hADSCs-exo into MIA-induced OA rats. Many studies have asserted, or at least theorized, that many patients do not respond to a single injection of hADSCs-exo. Furthermore, there is a greater risk of aneurysm formation in the unresponsive group than among those rats who defervesce completely after a single injection of hADSCs-exo. The limitations of the present study were as follows: the sample size was small, and further analysis with larger samples of other independent sets, as well as specific samples such as peripheral blood T cells, monocytes/macrophages, would be necessary to confirm the material effect of ahADSCs-exo injection.


  Conclusion Top


The present study investigated the effects of hADSCs-exo on OA pathology, differential gene expression, and the level of related proteins in MIA-induced OA in rats. The interaction network consisted of 57 proteins encoded by upregulated genes. However, the interaction network also consisted of 69 proteins encoded by downregulated genes [Figure 3]. The changes in gene expression suggest the potential biological pathway that occurs in the angiogenesis, inflammatory response, immune response, cell cycle, cell death, cell differentiation, cell proliferation, DNA repair, RNA splicing, and secretion. In addition, understanding the processes driving hADSCs-exo recruitment could prove invaluable in the development of novel, targeted therapies to selectively inhibit pathological responses in OA.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2017R1D1A1B03028112).

Financial support and sponsorship

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2017R1D1A1B03028112).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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