Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization
Abstract
:1. Introduction
2. Results
2.1. MSC Sheet Transplantation Accelerates Healing of Pressure Ulcer Defect
2.2. Cell Sheet Transplantation Induces Intensive Remodeling of Granulation Tissue
2.3. Evaluation of Transplant Retention after MSC Delivery by Suspension or Cell Sheets
2.4. Cell Sheets Promote Formation of Granulation Tissue at Early Stages of Healing
2.5. Vascularization of GT Is Modulated by MSC Secretome and CS
2.6. Assembly of MSC in Cell Sheets Increases Secretion of Growth Factors Involved in Blood Vessel Maturation
2.7. RNA-Sequencing of Cell Sheets Shows Significant Changes of MSC Transcriptome Profile Compared to Monolayer Culture
3. Discussion
4. Materials and Methods
4.1. Cell Cultures
4.2. MSC Secretome Preparation
4.3. Fabrication of Cell Sheets from MSC
4.4. BioPlex Assay of Growth Factors in MSC Monolayer and Cell Sheet Secretomes
4.5. RNA Isolation and Transcriptome RNA-Sequencing Analysis
4.6. Pressure Ulcer Model
4.7. Delivery of MSC Suspension, Secretome or MSC Sheets
- (1)
- “Suspension”—injection suspended MSC to the edges and bottom of skin defect.
- (2)
- “Secretome”—injection of MSC secretome into the edges and bottom of skin defect.
- (3)
- “Cell Sheet”—application of CS to ulcer surface.
- (4)
- “Untreated”—animals without therapy.
4.8. Wound Assessment and Histological Examination
4.9. Histological Evaluaton of Healing Processes: Granulation Tissue Formation and Maturation
4.10. Immunofluorescent Staining and Vessel Density Analysis
4.11. PKH26 Labelling and Subsequent Detection of Transplanted Cells in Histological Preparations
4.12. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Ang-1 | Angiopoietin-1 |
Ang-2 | Angiopoietin-2 |
ANOVA | Analysis of Variation |
αSMA | Alpha smooth muscle actin |
BP | Biological process |
CS | Cell sheet |
DEG | Differentially expressed genes |
DNA | Deoxyribonucleic acid |
ECM | Extracellular matrix |
ELISA | Enzyme-linked immunosorbent assay |
FBS | Fetal bovine serum |
FGF | Fibroblast growth factor |
GAPDH | Glyceraldehyde 3-phosphate dehydrogenase |
G-CSG | Granulocyte-colony stimulating factor |
GO | Gene ontology |
GT | Granulation tissue |
HGF | Hepatocyte growth factor |
LOX | Lysyl oxidase |
MF | Molecular function |
MSC | Mesenchymal stromal cell |
NFKB1 | Nuclear factor κB |
PDGF | Platelet-derived growth factor |
RELA | REL-associated protein |
RNA | Ribonucleic acid |
SP-1 | Specificity protein 1 |
TF | Transcription factor |
TGF | Transforming growth factor |
Tie-2 | Endothelial specific receptor tyrosine kinase |
VEGF | Vascular endothelial growth factor |
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Rank | TF | Overlapped Genes N | List of Overlapped Genes |
---|---|---|---|
1 | SP1 | 28 | C4A, AGTR1, EDNRB, AGT, PPL, MMP2, TNC, EGR1, LSP1, CCND2, TNFSF10, MME, FBLN1, CYP27A1, SOX9, COMP, SLC39A8, COL18A1, C4B, SREBF1, HGF, TCN2, ISG20, APOE, SOD2, CYP19A1, PTN, CHI3L1 |
2 | NFKB1 | 22 | EGR1, BGN, CYP19A1, PTGFR, TNC, SOD2, ADORA1, CXCL12, TNFSF13B, MMP2, CD74, FGF7, IRF4, AGT, SLC25A27, PLA2G2A, CCND2, BCL2L11, IRF7, VCAM1, A2M, TNFSF10 |
3 | RELA | 20 | IRF7, EGR1, FGF7, ADORA1, CXCL12, MMP2, SLC25A27, SOD2, VCAM1, BCL2L11, TNFSF10, PLA2G2A, TNC, CD74, CYP19A1, CCND2, PTGFR, AGT, BGN, IRF4 |
4 | FOXO3 | 8 | BCL6, TXNIP, TNFSF10, BCL2L11, CCND2, VEGFA, CDKN2B, VEGFB |
5 | USF1 | 8 | FMO3, TCN2, AGT, CYP19A1, SLC1A3,LIPC, CTSD, ISG20 |
6 | SREBF1 | 5 | LRP1, ACACB, LDLR, FASN, CIC |
7 | STAT3 | 10 | CCND2, CFB, PROS1, HGF, MMP2, CYP19A1, A2M, CHI3L1, DIRAS3, BCL6 |
8 | HIF1A | 12 | TGFB3, TLR6, VEGFA, ARNT, TIMP2, SOCS1, VEGFB, MMP2, CXCL12, EDNRB, ACE, AGTR1 |
9 | FOXO1 | 4 | TNFSF10, EGR1, ANGPT2, TXNIP |
10 | JUN | 10 | DCN, VCAM1, CYP19A1, MMP2, SOD2, PTN, TNC, MGP, FGF7, LBP |
11 | DNMT1 | 4 | ESR1, IL32, CDKN2B, VEGFA |
12 | SP3 | 8 | FBLN1, HGF, SLC1A3, ACE, TCN2, MMP2, AGTR1, CYP27A1 |
13 | CREB1 | 7 | NR4A3, AQP3, BCL2L11, BDKRB2, SOX9, MMP2, CYP19A1 |
14 | MYC | 7 | VEGFA, JUNB, HLA-B, TFAP4, SHMT1, MST1, BCL2 |
15 | ESR1 | 6 | ESR1, BCL2, VEGFA, ZEB1, CEBPB, JUNB |
16 | ETS2 | 4 | EGR1, ANGPT2, MMP2, TNC |
17 | WT1 | 5 | WTAP, BCL2, VEGFA, VEGFB, JUNB |
18 | ATF4 | 4 | IRF7, HRK, DDIT4, APOE |
19 | PPARA | 4 | TXNIP, SOD2, G0S2, CD36 |
20 | SPI1 | 5 | JCHAIN, BCL6, MME, CTSS, CTSK |
21 | MYCN | 4 | EFNB3, CTSD, MXI1, CLU |
22 | USF2 | 4 | TCN2, CTSD, CYP19A1, LIPC |
23 | HDAC1 | 5 | HLA-DRA, EGR1, CCND2, TXNIP, SFRP1 |
24 | STAT1 | 4 | UPP1, XAF1, STAT2, MUC1 |
25 | BRCA1 | 4 | ESR1, IRF9, VEGFA, DDIT3 |
26 | TP53 | 8 | BDKRB2, GPNMB, PDGFRB, AQP3, EGR1, PMAIP1, CTSD, MMP2 |
27 | ETS1 | 5 | DUSP6, ANGPT2, EGR1, TMEM158, TNC |
28 | YY1 | 5 | SAP30, VWF, LSS, VEGFB, LDLR |
29 | BRCA1 | 4 | EGR1, CTSD, IRF7, CYP19A1 |
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Alexandrushkina, N.; Nimiritsky, P.; Eremichev, R.; Popov, V.; Arbatskiy, M.; Danilova, N.; Malkov, P.; Akopyan, Z.; Tkachuk, V.; Makarevich, P. Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization. Int. J. Mol. Sci. 2020, 21, 5567. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155567
Alexandrushkina N, Nimiritsky P, Eremichev R, Popov V, Arbatskiy M, Danilova N, Malkov P, Akopyan Z, Tkachuk V, Makarevich P. Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization. International Journal of Molecular Sciences. 2020; 21(15):5567. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155567
Chicago/Turabian StyleAlexandrushkina, Natalya, Peter Nimiritsky, Roman Eremichev, Vladimir Popov, Mikhail Arbatskiy, Natalia Danilova, Pavel Malkov, Zhanna Akopyan, Vsevolod Tkachuk, and Pavel Makarevich. 2020. "Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization" International Journal of Molecular Sciences 21, no. 15: 5567. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21155567