Journal of Animal Reproduction and Biotechnology 2022; 37(4): 246-254
Published online December 31, 2022
https://doi.org/10.12750/JARB.37.4.246
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Seung-Chan Lee1,# , Chae-Yeon Hong2,#
, Yong-Ho Choe2
, Tae-Seok Kim2
, Won-Jae Lee3
, Gyu-Jin Rho2,4
and Sung-Lim Lee2,4,*
1Apures, Inc., Central Research Center, Pyeongtaek 17792, Korea
2College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea
3College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
4Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea
Correspondence to: Sung-Lim Lee
E-mail: sllee@gnu.ac.kr
#These authors contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/ drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and antiinflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient’s synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RAhMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.
Keywords: 3D-spheroid, immunomodulation, mesenchymal stem cell, rheumatoid arthritis, synovial fluid
Mesenchymal stem cells (MSCs) are of great interest for the use in regenerative medicine, since they have capabilities of multi-lineage differentiation, immunomodulation, and secretion of pro-regenerative factors. MSCs has an ability of immunomodulation through cell to cell contacts or secretion of soluble factors, making them ideal candidates for cell therapy to treat certain kinds of immune-mediated disorders (Nauta and Fibbe, 2007; Zhao et al., 2010; Petrie Aronin and Tuan, 2010). Therefore, the mechanisms through which MSCs exert their immunomodulatory functions in autoimmune diseases have been studied (Djouad et al., 2005; Augello et al., 2007).
MSCs can be easily isolated from RA patient’s synovial fluid, and they can be obtained during diagnosis or treatment of patients when clinicians confirm a diagnosis of RA without harming (Mochizuki et al., 2006). Moreover, synovial fluid is an important source of MSC for the immoderation therapy of rheumatoid arthritis (RA), facilitating systemic recovery from RA by regenerating cartilage or bone repair in damaged RA joints. However, there is a dramatic increase in cellular senescence in MSCs derived from synovial fluid of longstanding RA patients, which may induce the lost immunomodulatory properties of those MSCs (Lee et al., 2021). Therefore, it is necessary to improve the immunomodulatory property of MSC derived from RA patients with reduced this function. Recently, studies of three-dimensional culture systems have been conducted for the effective application by enhancing the potential of MSCs as we mimick the
In the present study, we monitored morphological and cellular changes of MSCs derived from synovial fluid of longstanding RA joints over 13 years (RA-hMSCs) under 3D-cuture condition and the evaluated mRNA expression levels of anti-inflammatory and anti-tumorigenic molecules following exposure to RA synovial fluid.
All media and chemicals were purchased from Gibco (Invitrogen Corporation, Grand Island, NY, USA) and Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise specified. The MSC culture medium was advanced Dulbecco’s modified Eagle medium (ADMEM), supplemented with 10% of fetal bovine serum (FBS), 1% GlutaMax (Gibco), 10 ng/mL of basic fibroblast growth factor (bFGF), and 1.0% penicillin-streptomycin (10,000 IU and 10,000 μg/mL, respectively, Pen-Strep), The osmolarity was adjusted to 285 ± 5 mOsm/kg, and the pH was adjusted to 7.2.
For this study, we used two different cell lines, RA-hMSCs-1 and -2, which we used in our previous report (Lee et al., 2021). In briefly, RA-hMSCs were isolated from SF from the femorotibial joint of RA patients with chronic inflammation (average 13.8 years) and authorized by the IRB (Approval number GNUH 2012-05-009) at Hospital of Gyeongsang National University. We performed all experiments using both cell lines in consideration of differences between patient-derived cell lines.
RA-hMSCs were confirmed maintain characterics of MSC by MSC-specific cell surface markers of RA-hMSCs were analyzed by flow cytometry analysis (BD FACS Calibur; Becton Dickinson Franklin Lakes, NJ, USA). A total of 1 × 104 cells were harvested and fixed with 4% paraformaldehyde at 4℃. All antibodies were diluted (1:200) with 1% bovine serum albumin. Fluorescein isothiocyanate (FITC)-conjugated primary antibodies were incubated with the harvested cells for 1 h, with mouse IgG1-FITC used as an isotype control.
Approximately ~80% of confluent RA-hMSCs are differentiated into adipocytes and osteoblasts after 3 weeks. Adipogenesis was induced with Advanced Dulbecco’s modified Eagle’s medium (ADMEM) supplemented with 10% FBS, 10 µM insulin, 200 µM indomethacin, 500 µM isobutyl methylxanthine, and 1 µM dexamethasone for 28 d. Then, adipogenesis was confirmed by intracellular lipid vacuole staining with 0.5% Oil red O solution. And, osteogenesis was induced with ADMEM containing 10% FBS, 1 µM dexamethasone, 10 mM sodium β-glycerophosphate and 100 µM ascorbic acid for 28 d. Then, osteogenesis was determined by the accumulation of calcium deposits visualized with Alizarin-red S solution.
To evaluate the cell proliferation capacity, ~1 × 103 cells at passage 3-5 were suspended in 500 μL of ADMEM, supplemented with 10% FBS and cultured for 14 d, and the culture medium was changed every 3 d. Cells were harvested with 0.25% trypsin EDTA every 2 d and counted in triplicate with flow cytometry.
RA-hMSCs were plated as hanging drops on an inverted culture dish lid in 30µL of ADMEM containing 25,000 cells/drop. The lid was flipped and placed on a culture dish containing PBS to prevent evaporation. Hanging drop cultures were grown at 37℃ up to 3 d in a humidified atmosphere with 5% CO2.
2D-monolayer cells were cultured in ADMEM supplemented with 20% synovial fluid of longstanding RA patients for 48 h. And 3D-spheroid cells were cultured in ADMEM supplemented with 20% synovial fluid of longstanding RA patients for 48 h using low attachment plates.
Total RNAs were extracted by using RNeasy mini Kit (Qiagen) and quantified by using the OPTIZEN 3220 UV BIO Spectrophotometer. Reverse transcription of 1 µg total RNAs were performed by using Omniscript Reverse Transcription Kit (Qiagen) with oligo dT primer (Invitrogen) at 60℃ for 1 h.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used for gene expression studies to determine pluripotency (
We evaluated anti-inflammatory and anti-tumorigenic genes. qRT-PCR was performed in triplicate for
Table 1 . qRT-PCR primer sequence of anti-inflammatory and anti-tumorigenic-related genes
Target Gene | Sequence | Accession number | Base pair |
---|---|---|---|
F: CGAAGCTGAATCAGGAGAAG R: TTTGTGGGACAGCATGGTG | NM_003406.3 | 111 | |
F: CAGGCTTCCCAAATGAGTACGA R: CCTGGGTCATCTTCAAGGTCAA | NM_007115.3 | 115 | |
F: GGTGCTCCACTTTCCAAAGGAT R: CTCAGTGATGGCTTCAGGGTTC | NM_003155.2 | 104 | |
F: ATCGTCCTCCTTGTCCCTGACT R: AGCTCCAACCCCACACACTATG | NM_002309.4 | 105 | |
F: GCTCTGGCTGAACTTTGTGG R: TCTGCAATAGCACTTATGTCATGG | NM_006850.3 | 133 | |
F: CAGTAGTAGCCTCCAGGTTTCC R: CTCGTGATCTACCCACCTTGG | NM_003810.3 | 148 | |
F: TCCTGGAAATCATCAAGCAAGG R: GGCTCCAAGGAAAGCATAGAGG | NM_001008540.1 | 125 | |
F: TGGAGAAGGTCTGTCTTGCC R: GGAGTTCACTGCATTTGGATAGC | NM_012242.2 | 112 |
One-way analysis of variance (ANOVA) with Tukey’s posthoc test was used to analyze differences among the treatments using SPSS software. Data is expressed as the mean ± standard deviation (SD), and differences were considered significant when
Two different cell lines of hMSCs derived from longstanding RA-patients, RA-hMSCs-1 and -2 were maintained morphology of fibroblastic morphologies with plastic attachment ability and cell surface markers for RA-hMSCs were identified with flow cytometry using a BD FACS Calibur instrument (Fig. 1A). In briefly, RA-hMSCs-1 and -2 were identified in cells negative for hematopoietic stem cell markers (CD34 and CD45) and positive for MSC-specific markers (CD44, CD90 and CD105).
RA-hMSCs-1 and -2 were verified by cytochemical staining from both sources progressed toward differentiation into adipocytes and osteocytes (Fig. 1B). It is confirmed by the cytoplasmic accumulation of lipid vacuoles and the deposition of calcified extracellular matrix by Oil red O and Alizarin red S staining.
We assessed morphology change of RA synovial fluid (RA-SF) non-treated RA-hMSCs as control and RA-SF treated hMSCs for 48 h. In non-treated control RA-hMSCs showed fibroblast-like morphology, but cellular debris was increased within the media of RA-hMSCs following treatment of RA-SF (Fig. 2).
RA-hMSCs were generated spheroids using StemFIT culture dishes and cultured in hanging drops formed a loose network after 24 h. At 48 h, numerous small aggregates gradually were coalesced into a single central spheroid along the lower surface of the drop. Once spheroids are formed, it lasted up to 72 h (Fig. 3A). We re-isolated spheroid-forming RA-hMSCs into single cells by trypsin-EDTA treatment. The difference of 2D-monolayer and 3D-spheroid cultured hMSCs size was estimated by microscopy (Fig. 3B). The cells are released from spheroids after trypsinization and then the cells were estimated that they are nearly half the diameter and approximately one-fourth the volume of RA-hMSCs derived from 2D cultured. The mRNA levels of pluripotency related genes (
We measured mRNA levels of anti-inflammatory genes (
However, the expression patterns of DKK1 was observed cell line dependent difference between both RA-hMSCs. In RA-hMSCs-1, the mRNA level of DKK1 was significantly (
As autoimmune disease, RA therapy with MSC displays an improved multilineage differentiation and immunomodulation potential that repairs not only destructed cartilage or bones in affected joints but also ameliorates the autologous immune response to chronic systemic inflammation. SF-MSCs can be easily obtained as a cell source for stem cell treatment by aspirating SF with a needle and syringe from the joints of patients diagnosed as RA (Mochizuki et al., 2006; Jones et al., 2008; Sekiya et al., 2012). Up to this time, the immunosuppression potential of MSCs derived from synovial sources in the treatment of RA has not been investigated. Most studies that have investigated SF-MSCs have analyzed characterization of cells derived from OA or injured joints in patients who may have each different physiological case (Jones et al., 2008; Sekiya et al., 2012).
However, the incidence of RA increases rapidly in aged women who are at onset of the menopause (Talsania and Schofield, 2017). In our pervious study, longstanding RA patient derived MSCs lost immunomodulatory property and increased cellular senescence with chronic inflammation (Lee et al., 2021), so these patient-derived MSCs require stemness recovery for improvement of immunomodulation property. Therefore, we established 3D-spheroid forming system of cellular senescence increased RA-hMSCs derived from longstanding RA patients for increasing of ant-inflammation and anti-tumorigenic related genes.
MSCs dissociated from spheroids provide extremely small activated cells that could have major advantages for i.v. administration. As cells cultured with 2D did not show capacity of supporting a proper microenvironment for them (Bara et al., 2014), a microenvironment of conditions of being cultured is important for the decision of cell fate (Mason et al., 2011; Tsai et al., 2015). Thus, many studies have focused on 3D culture system of MSCs to maintain and enhance stemness along with pluripotent characteristics of MSCs (Bartosh et al., 2010). MSCs aggregate with each other in 3D culture conditions to form efficient spheroids and have cellular niches similar to
In the present study, we successfully generated RA-hMSCs spheroids using StemFIT culture dishes and keep cultured in hanging drops formed for 72 h. These spheroid-formed cells were re-isolated after 72 h, reduced in size and changed to more round intact shape than in 2D-monolyaere culture. Morphological changes in cells to large and irregular shapes mean that the quality of cells is poor. Moreover, the size of MSCs has been critically considered in clinical applications after intravascular injection (i.v.) (Ge et al., 2014). The size of the i.v. injected MSCs should be smaller than the inner diameter of the peripheral vessels to prevent significant vascular occlusion, which can have serious consequences.
The 3D-spheroid formation of MSCs by aggregation is one of the useful methods to enhance the valuable therapeutic potential in terms of enhancement of stemness, immunomodulatory, and anti-inflammatory properties, and chemotaxis for homing (Jauković et al., 2020). Many different methods have been reported for preparing MSC spheroids, including hanging drop (Huang et al., 2020), concave micro-well aggregation (Hsu et al., 2013; Lee et al., 2016) and gravity circulation (Imura et al., 2018) methods. The expression levels of
Based on these results, we hypothesize that RA-hMSCs can be activated non-chemically in hanging drops to increase expression levels of pluripotent related genes, anti-inflammatory genes and anti-tumorigenic genes. Therefore, the 3D-spheroid RA-hMSCs may have advantages for therapeutic applications in RA using MSCs derived from longstanding patients.
None.
Conceptualization, S-C.L., C-Y.H.; data curation, S-C.L., C-Y.H.; formal analysis, Y-H.C.; investigation, S-C.L., C-Y.H., W-J.L., T-S.K.; methodology, S-C.L., C-Y.H., W-J.L.; project administration, S-L.L.; resources, S-L.L., G-J.R.; supervision, G-J.R.; writing - original draft, S-C.L., C-Y.H.; writing - review & editing, S-L.L.
This study was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (grant no. NRF-2021R1A2C1007054).
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Journal of Animal Reproduction and Biotechnology 2022; 37(4): 246-254
Published online December 31, 2022 https://doi.org/10.12750/JARB.37.4.246
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Seung-Chan Lee1,# , Chae-Yeon Hong2,#
, Yong-Ho Choe2
, Tae-Seok Kim2
, Won-Jae Lee3
, Gyu-Jin Rho2,4
and Sung-Lim Lee2,4,*
1Apures, Inc., Central Research Center, Pyeongtaek 17792, Korea
2College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Korea
3College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea
4Research Institute of Life Sciences, Gyeongsang National University, Jinju 52828, Korea
Correspondence to:Sung-Lim Lee
E-mail: sllee@gnu.ac.kr
#These authors contributed equally to this work.
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Current studies have revealed the capacity of mesenchymal stem cells (MSCs) in term of immunomodulatory properties, and this distinct potential is downgraded according to the disease duration of patients-derived MSCs. In order to enhance the immunomodulatory and anti-tumorigenic properties of the rheumatoid arthritis (RA) joints-derived MSCs, we aggregate synovial fluid-derived MSCs from RA joints (RA-hMSCs) into 3D-spheroids by the use of hanging drop culture method. Cells were isolated from synovial fluids of RA joints with longstanding active status over 13 years. For aggregation of RA-hMSCs into 3D-spheroids, cells were plated in hanging drops in 30 μL of advanced DMEM (ADMEM) containing 25,000-30,000 cells/ drop and cultured for 48 h. To analyze the comparative immunomodulatory effects of 3D-spheroid and 2D monolayer cultured RA-hMSCs and then cells were cultured in ADMEM supplemented with 20% of synovial fluids of RA patients for 48 h and were evaluated by qRT-PCR for their expression of mRNA levels of inflammatory and antiinflammatory markers. Cellular aggregation of RA-hMSCs was observed and cells were aggregate into a single sphere. Following treatment of RA patient’s synovial fluids into the RA-hMSCs, spheroids formed RA-hMSCs showed significantly (p < 0.05) higher expression of TNFα stimulated gene/protein 6 (TSG-6) than the monolayer cultured RAhMSCs. Therefore, the 3D-spheroid culture methods of RA-hMSCs were more effective than 2D monolayer cultures in suppressing inflammatory response treated with 20% of RA-synovial fluids by expression of TNFα (TSG-6) according to the immune response and enhanced secretion of inflammatory factors.
Keywords: 3D-spheroid, immunomodulation, mesenchymal stem cell, rheumatoid arthritis, synovial fluid
Mesenchymal stem cells (MSCs) are of great interest for the use in regenerative medicine, since they have capabilities of multi-lineage differentiation, immunomodulation, and secretion of pro-regenerative factors. MSCs has an ability of immunomodulation through cell to cell contacts or secretion of soluble factors, making them ideal candidates for cell therapy to treat certain kinds of immune-mediated disorders (Nauta and Fibbe, 2007; Zhao et al., 2010; Petrie Aronin and Tuan, 2010). Therefore, the mechanisms through which MSCs exert their immunomodulatory functions in autoimmune diseases have been studied (Djouad et al., 2005; Augello et al., 2007).
MSCs can be easily isolated from RA patient’s synovial fluid, and they can be obtained during diagnosis or treatment of patients when clinicians confirm a diagnosis of RA without harming (Mochizuki et al., 2006). Moreover, synovial fluid is an important source of MSC for the immoderation therapy of rheumatoid arthritis (RA), facilitating systemic recovery from RA by regenerating cartilage or bone repair in damaged RA joints. However, there is a dramatic increase in cellular senescence in MSCs derived from synovial fluid of longstanding RA patients, which may induce the lost immunomodulatory properties of those MSCs (Lee et al., 2021). Therefore, it is necessary to improve the immunomodulatory property of MSC derived from RA patients with reduced this function. Recently, studies of three-dimensional culture systems have been conducted for the effective application by enhancing the potential of MSCs as we mimick the
In the present study, we monitored morphological and cellular changes of MSCs derived from synovial fluid of longstanding RA joints over 13 years (RA-hMSCs) under 3D-cuture condition and the evaluated mRNA expression levels of anti-inflammatory and anti-tumorigenic molecules following exposure to RA synovial fluid.
All media and chemicals were purchased from Gibco (Invitrogen Corporation, Grand Island, NY, USA) and Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise specified. The MSC culture medium was advanced Dulbecco’s modified Eagle medium (ADMEM), supplemented with 10% of fetal bovine serum (FBS), 1% GlutaMax (Gibco), 10 ng/mL of basic fibroblast growth factor (bFGF), and 1.0% penicillin-streptomycin (10,000 IU and 10,000 μg/mL, respectively, Pen-Strep), The osmolarity was adjusted to 285 ± 5 mOsm/kg, and the pH was adjusted to 7.2.
For this study, we used two different cell lines, RA-hMSCs-1 and -2, which we used in our previous report (Lee et al., 2021). In briefly, RA-hMSCs were isolated from SF from the femorotibial joint of RA patients with chronic inflammation (average 13.8 years) and authorized by the IRB (Approval number GNUH 2012-05-009) at Hospital of Gyeongsang National University. We performed all experiments using both cell lines in consideration of differences between patient-derived cell lines.
RA-hMSCs were confirmed maintain characterics of MSC by MSC-specific cell surface markers of RA-hMSCs were analyzed by flow cytometry analysis (BD FACS Calibur; Becton Dickinson Franklin Lakes, NJ, USA). A total of 1 × 104 cells were harvested and fixed with 4% paraformaldehyde at 4℃. All antibodies were diluted (1:200) with 1% bovine serum albumin. Fluorescein isothiocyanate (FITC)-conjugated primary antibodies were incubated with the harvested cells for 1 h, with mouse IgG1-FITC used as an isotype control.
Approximately ~80% of confluent RA-hMSCs are differentiated into adipocytes and osteoblasts after 3 weeks. Adipogenesis was induced with Advanced Dulbecco’s modified Eagle’s medium (ADMEM) supplemented with 10% FBS, 10 µM insulin, 200 µM indomethacin, 500 µM isobutyl methylxanthine, and 1 µM dexamethasone for 28 d. Then, adipogenesis was confirmed by intracellular lipid vacuole staining with 0.5% Oil red O solution. And, osteogenesis was induced with ADMEM containing 10% FBS, 1 µM dexamethasone, 10 mM sodium β-glycerophosphate and 100 µM ascorbic acid for 28 d. Then, osteogenesis was determined by the accumulation of calcium deposits visualized with Alizarin-red S solution.
To evaluate the cell proliferation capacity, ~1 × 103 cells at passage 3-5 were suspended in 500 μL of ADMEM, supplemented with 10% FBS and cultured for 14 d, and the culture medium was changed every 3 d. Cells were harvested with 0.25% trypsin EDTA every 2 d and counted in triplicate with flow cytometry.
RA-hMSCs were plated as hanging drops on an inverted culture dish lid in 30µL of ADMEM containing 25,000 cells/drop. The lid was flipped and placed on a culture dish containing PBS to prevent evaporation. Hanging drop cultures were grown at 37℃ up to 3 d in a humidified atmosphere with 5% CO2.
2D-monolayer cells were cultured in ADMEM supplemented with 20% synovial fluid of longstanding RA patients for 48 h. And 3D-spheroid cells were cultured in ADMEM supplemented with 20% synovial fluid of longstanding RA patients for 48 h using low attachment plates.
Total RNAs were extracted by using RNeasy mini Kit (Qiagen) and quantified by using the OPTIZEN 3220 UV BIO Spectrophotometer. Reverse transcription of 1 µg total RNAs were performed by using Omniscript Reverse Transcription Kit (Qiagen) with oligo dT primer (Invitrogen) at 60℃ for 1 h.
Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used for gene expression studies to determine pluripotency (
We evaluated anti-inflammatory and anti-tumorigenic genes. qRT-PCR was performed in triplicate for
Table 1. qRT-PCR primer sequence of anti-inflammatory and anti-tumorigenic-related genes.
Target Gene | Sequence | Accession number | Base pair |
---|---|---|---|
F: CGAAGCTGAATCAGGAGAAG R: TTTGTGGGACAGCATGGTG | NM_003406.3 | 111 | |
F: CAGGCTTCCCAAATGAGTACGA R: CCTGGGTCATCTTCAAGGTCAA | NM_007115.3 | 115 | |
F: GGTGCTCCACTTTCCAAAGGAT R: CTCAGTGATGGCTTCAGGGTTC | NM_003155.2 | 104 | |
F: ATCGTCCTCCTTGTCCCTGACT R: AGCTCCAACCCCACACACTATG | NM_002309.4 | 105 | |
F: GCTCTGGCTGAACTTTGTGG R: TCTGCAATAGCACTTATGTCATGG | NM_006850.3 | 133 | |
F: CAGTAGTAGCCTCCAGGTTTCC R: CTCGTGATCTACCCACCTTGG | NM_003810.3 | 148 | |
F: TCCTGGAAATCATCAAGCAAGG R: GGCTCCAAGGAAAGCATAGAGG | NM_001008540.1 | 125 | |
F: TGGAGAAGGTCTGTCTTGCC R: GGAGTTCACTGCATTTGGATAGC | NM_012242.2 | 112 |
One-way analysis of variance (ANOVA) with Tukey’s posthoc test was used to analyze differences among the treatments using SPSS software. Data is expressed as the mean ± standard deviation (SD), and differences were considered significant when
Two different cell lines of hMSCs derived from longstanding RA-patients, RA-hMSCs-1 and -2 were maintained morphology of fibroblastic morphologies with plastic attachment ability and cell surface markers for RA-hMSCs were identified with flow cytometry using a BD FACS Calibur instrument (Fig. 1A). In briefly, RA-hMSCs-1 and -2 were identified in cells negative for hematopoietic stem cell markers (CD34 and CD45) and positive for MSC-specific markers (CD44, CD90 and CD105).
RA-hMSCs-1 and -2 were verified by cytochemical staining from both sources progressed toward differentiation into adipocytes and osteocytes (Fig. 1B). It is confirmed by the cytoplasmic accumulation of lipid vacuoles and the deposition of calcified extracellular matrix by Oil red O and Alizarin red S staining.
We assessed morphology change of RA synovial fluid (RA-SF) non-treated RA-hMSCs as control and RA-SF treated hMSCs for 48 h. In non-treated control RA-hMSCs showed fibroblast-like morphology, but cellular debris was increased within the media of RA-hMSCs following treatment of RA-SF (Fig. 2).
RA-hMSCs were generated spheroids using StemFIT culture dishes and cultured in hanging drops formed a loose network after 24 h. At 48 h, numerous small aggregates gradually were coalesced into a single central spheroid along the lower surface of the drop. Once spheroids are formed, it lasted up to 72 h (Fig. 3A). We re-isolated spheroid-forming RA-hMSCs into single cells by trypsin-EDTA treatment. The difference of 2D-monolayer and 3D-spheroid cultured hMSCs size was estimated by microscopy (Fig. 3B). The cells are released from spheroids after trypsinization and then the cells were estimated that they are nearly half the diameter and approximately one-fourth the volume of RA-hMSCs derived from 2D cultured. The mRNA levels of pluripotency related genes (
We measured mRNA levels of anti-inflammatory genes (
However, the expression patterns of DKK1 was observed cell line dependent difference between both RA-hMSCs. In RA-hMSCs-1, the mRNA level of DKK1 was significantly (
As autoimmune disease, RA therapy with MSC displays an improved multilineage differentiation and immunomodulation potential that repairs not only destructed cartilage or bones in affected joints but also ameliorates the autologous immune response to chronic systemic inflammation. SF-MSCs can be easily obtained as a cell source for stem cell treatment by aspirating SF with a needle and syringe from the joints of patients diagnosed as RA (Mochizuki et al., 2006; Jones et al., 2008; Sekiya et al., 2012). Up to this time, the immunosuppression potential of MSCs derived from synovial sources in the treatment of RA has not been investigated. Most studies that have investigated SF-MSCs have analyzed characterization of cells derived from OA or injured joints in patients who may have each different physiological case (Jones et al., 2008; Sekiya et al., 2012).
However, the incidence of RA increases rapidly in aged women who are at onset of the menopause (Talsania and Schofield, 2017). In our pervious study, longstanding RA patient derived MSCs lost immunomodulatory property and increased cellular senescence with chronic inflammation (Lee et al., 2021), so these patient-derived MSCs require stemness recovery for improvement of immunomodulation property. Therefore, we established 3D-spheroid forming system of cellular senescence increased RA-hMSCs derived from longstanding RA patients for increasing of ant-inflammation and anti-tumorigenic related genes.
MSCs dissociated from spheroids provide extremely small activated cells that could have major advantages for i.v. administration. As cells cultured with 2D did not show capacity of supporting a proper microenvironment for them (Bara et al., 2014), a microenvironment of conditions of being cultured is important for the decision of cell fate (Mason et al., 2011; Tsai et al., 2015). Thus, many studies have focused on 3D culture system of MSCs to maintain and enhance stemness along with pluripotent characteristics of MSCs (Bartosh et al., 2010). MSCs aggregate with each other in 3D culture conditions to form efficient spheroids and have cellular niches similar to
In the present study, we successfully generated RA-hMSCs spheroids using StemFIT culture dishes and keep cultured in hanging drops formed for 72 h. These spheroid-formed cells were re-isolated after 72 h, reduced in size and changed to more round intact shape than in 2D-monolyaere culture. Morphological changes in cells to large and irregular shapes mean that the quality of cells is poor. Moreover, the size of MSCs has been critically considered in clinical applications after intravascular injection (i.v.) (Ge et al., 2014). The size of the i.v. injected MSCs should be smaller than the inner diameter of the peripheral vessels to prevent significant vascular occlusion, which can have serious consequences.
The 3D-spheroid formation of MSCs by aggregation is one of the useful methods to enhance the valuable therapeutic potential in terms of enhancement of stemness, immunomodulatory, and anti-inflammatory properties, and chemotaxis for homing (Jauković et al., 2020). Many different methods have been reported for preparing MSC spheroids, including hanging drop (Huang et al., 2020), concave micro-well aggregation (Hsu et al., 2013; Lee et al., 2016) and gravity circulation (Imura et al., 2018) methods. The expression levels of
Based on these results, we hypothesize that RA-hMSCs can be activated non-chemically in hanging drops to increase expression levels of pluripotent related genes, anti-inflammatory genes and anti-tumorigenic genes. Therefore, the 3D-spheroid RA-hMSCs may have advantages for therapeutic applications in RA using MSCs derived from longstanding patients.
None.
Conceptualization, S-C.L., C-Y.H.; data curation, S-C.L., C-Y.H.; formal analysis, Y-H.C.; investigation, S-C.L., C-Y.H., W-J.L., T-S.K.; methodology, S-C.L., C-Y.H., W-J.L.; project administration, S-L.L.; resources, S-L.L., G-J.R.; supervision, G-J.R.; writing - original draft, S-C.L., C-Y.H.; writing - review & editing, S-L.L.
This study was supported by a grant from the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (grant no. NRF-2021R1A2C1007054).
Not applicable.
Not applicable.
Not applicable.
Not applicable.
No potential conflict of interest relevant to this article was reported.
Table 1 . qRT-PCR primer sequence of anti-inflammatory and anti-tumorigenic-related genes.
Target Gene | Sequence | Accession number | Base pair |
---|---|---|---|
F: CGAAGCTGAATCAGGAGAAG R: TTTGTGGGACAGCATGGTG | NM_003406.3 | 111 | |
F: CAGGCTTCCCAAATGAGTACGA R: CCTGGGTCATCTTCAAGGTCAA | NM_007115.3 | 115 | |
F: GGTGCTCCACTTTCCAAAGGAT R: CTCAGTGATGGCTTCAGGGTTC | NM_003155.2 | 104 | |
F: ATCGTCCTCCTTGTCCCTGACT R: AGCTCCAACCCCACACACTATG | NM_002309.4 | 105 | |
F: GCTCTGGCTGAACTTTGTGG R: TCTGCAATAGCACTTATGTCATGG | NM_006850.3 | 133 | |
F: CAGTAGTAGCCTCCAGGTTTCC R: CTCGTGATCTACCCACCTTGG | NM_003810.3 | 148 | |
F: TCCTGGAAATCATCAAGCAAGG R: GGCTCCAAGGAAAGCATAGAGG | NM_001008540.1 | 125 | |
F: TGGAGAAGGTCTGTCTTGCC R: GGAGTTCACTGCATTTGGATAGC | NM_012242.2 | 112 |
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pISSN: 2671-4639
eISSN: 2671-4663