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Cell Biosci. 2021;11(1):120.

Discovery of chemerin as the new chemoattractant of human mesenchymal stem cells.

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제1저자 김아이린, 박효민 · 교신저자 이은주, 김효수

PMID 34210352 PMC8252297 DOI 10.1186/s13578-021-00631-3 IF 6.5 순환기내과
소속 (5)
  1. Molecular Medicine & Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine, Seoul National University, Seoul National University Hospital, 101 DeaHak-ro, JongRo-gu, Seoul, 03080, Republic of Korea.
  2. Program in Stem Cell Biology, Seoul National University College of Medicine, Seoul, Republic of Korea.
  3. Biomedical Research Institute, Seoul National University Hospital, 101 DeaHak-ro, JongRo-gu, Seoul, 03080, Republic of Korea. leeunju@snu.ac.kr.
  4. Molecular Medicine & Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine, Seoul National University, Seoul National University Hospital, 101 DeaHak-ro, JongRo-gu, Seoul, 03080, Republic of Korea. usahyosoo@gmail.com.
  5. Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea. usahyosoo@gmail.com.

Abstract

BACKGROUND The homing capacity of human mesenchymal stem cells (hMSCs) to the injured sites enables systemic administration of hMSCs in clinical practice. In reality, only a small proportion of MSCs are detected in the target tissue, which is a major bottleneck for MSC-based therapies. We still don't know the mechanism how MSCs are chemo-attracted to certain target organ and engrafted through trans-endothelial migration. In this study, we aimed to determine the mechanism how the circulating hMSCs home to the injured liver.

METHODS AND RESULTS When we compare the cytokine array between normal and injured mouse liver at 1-day thioacetamide (TAA)-treatment, we found that chemerin, CXCL2, and CXCL10 were higher in the injured liver than normal one. Among three, only chemerin was the chemoattractant of hMSCs in 2D- and 3D-migration assay. Analysis of the signal transduction pathways in hMSCs showed that chemerin activated the phosphorylation of JNK1/2, ERK1/2 and p38, and finally upregulated CD44, ITGA4, and MMP-2 that are involved in the transendothelial migration and extravasation of MSCs. Upstream transcription regulators of CD44, ITGA4, and MMP-2 after chemerin treatment were MZF1, GATA3, STAT3, and STAT5A. To develop chemerin as a chemoattractant tool, we cloned gene encoding the active chemerin under the CMV promoter (CMV-aChemerin). We analyzed the migration of hMSCs in the 3D model for space of the Disse, which mimics transmigration of hMSCs in the liver. CMV-aChemerin-transfected hepatocytes were more effective to attract hMSC than control hepatocytes, leading to the enhanced transendothelial migration and homing of hMSCs to liver. The homing efficiency of the intravascularly-delivered hMSCs to liver was evaluated after systemic introduction of the CMV-aChemerin plasmid packed in liposome-vitamin A conjugates which target liver. CMV-aChemerin plasmid targeting liver significantly enhanced homing efficiency of hMSCs to liver compared with control plasmid vector.

CONCLUSIONS Chemerin is the newly found chemoattractant of hMSCs and may be a useful tool to manipulate the homing of the intravascularly-administered hMSC to the specific target organ.

Keywords

Active chemerinChemoattractantHoming of stem cellsIntravascularly-delivery

Figures

Fig. 1
Fig. 1 Screening of chemoattractant candidates for hMSCs. A Liver fibrosis model was generated by injecting TAA (0.2 mg/mL) every 2 days to the mouse. DiI-labeled hE-MSCs were systemically administered after the first TAA injection. DiI-labeled hE-MSCs were found in mouse liver treated with TAA. DiI hE-MSCs are red and the nuclei are blue. B Mouse chemokine array blots of normal and the injured liver at 1-day after TAA-treatment. The upper blot was exposed for 5 min and the bottom blot was exposed for 10 min. Chemokines are shown as duplicate dots. Reference dots are shown at the periphery; top right and left as well as bottom left. White squares indicate chemokines that were significantly increased in the injured mouse liver with TAA (0.2 mg/mL). The data shown were reproducible results from two independent experiments. Blots were quantified by Image J software. (C) 3D transmigration assay. Upper panel shows the scheme. The 3D transmigration system constructed using DiO-labeled HUVECs (green) as the endothelial layer on the surface of collagen gel below containing a given chemokine such as CXCL2, CXCL10, or chemerin (all at 50 pM). DiI-labeled hE-MSCs (red) were seeded on HUVECs and their migration into collagen gel was observed for 72 h by performing Z-stacking in a confocal microscope. Migration distances of hE-MSCs are indicated on a scale of 40 µm. The data shown are reproducible results from two independent experiments. D 2D random migration assay of hBM-MSC in response to chemerin or ChemR23 blocker (ChemR23 neutralizing antibody). Quantification of cell migration at 12 and 24 h was represented as the percentage of cell coverage in the initial cell-free zone. The white bar represents control hBM-MSCs, the gray bar indicates hBM-MSCs exposed to chemerin in media, and the black bar indicates hBM-MSCs that were treated with the ChemR23 neutralizing antibody in addition to chemerin
Fig. 2
Fig. 2 Chemerin induced phosphorylation of JNK1/2, ERK1/2, and p38 and increased the expression of migration-related genes in hBM-MSCs. A Western blot of phosphorylated JNK1/2, p38, and ERK1/2 in hBM-MSCs after chemerin treatment. Protein expression was quantified using the Image J software. Expression was normalized to that of GAPDH (N > 3). B RT-qPCR of CD44, ITGA4, and MMP-2 after chemerin treatment. mRNA levels were observed 24 h after the chemerin treatment of BM-MSCs. C Western blot analysis of CD44, ITGA4, and MMP-2 of hBM-MSCs after chemerin treatment. Protein expression was quantified using Image J and normalized to GAPDH level. D Venn diagram of common transcription factors, CD44, ITGA4, and MMP2. Transcription of each gene was found using GP miner and 26 genes were found to encode common transcription factors. Among these transcription factors, five were involved in transmigration. GP miner (http://gpminer.mbc.nctu.edu.tw/). E RT-qPCR of transcription factors in naïve and chemerin-treated hBM-MSCs. The data shown were reproducible results from three independent experiments
Fig. 3
Fig. 3 Construction of active chemerin vector. A Scheme showing the active chemerin vector. Twenty amino acids at N-terminus represent the signaling peptide that assists secretion of chemerin from hMSCs. The last six amino acids at C-terminus are enzymatically cleaved away leaving active chemerin. B Scheme for 3D mimesis of the space of Disse. The upper panel shows the possibility of transendothelial migration of hBM-MSCs in the space of Disse. To observe the possibility of ex vivo migration, a similar environment was constructed using Hepa-1c1c7 (hepatocytes), MS-1 (endothelial cells), and hBM-MSCs. C Evaluation of the vector in Hepa-1c1c7. Levels of the vector-encoded protein in the lysate and media of Hepa-1c1c7 cells transfected with the vector were detected using western blotting. The cell lysate shows different-sized chemerin and the supernatant shows inactive and active chemerin. Recombinant mouse chemerin (rmChemerin) was loaded as the positive control. D Transendothelial migration of hBM-MSCs. Penetration of hBM-MSCs through MS-1 cells into collagen matrix containing Hepa-1c1c7 cells transfected with active chemerin vector. MS-1 was labeled with DiO (green) and hBM-MSC was labeled with DiI (red). Migration in the vertical side of the collagen matrix was observed using the Z stack of the confocal microscope. The migration distances of hBM-MSCs were indicated in the scale of 50 µM. The data shown were reproducible results from two independent experiments
Fig. 4
Fig. 4 Applicability of the active chemerin vector in vivo. A Confocal microscopy images of DiI-labeled hBM-MSCs that homed to liver after systemic administration in mouse. Homing to liver was enhanced by pretreatment of pCMV-aChemerin vector in liposome-vitamin A conjugate. Red indicates DiI-labeled hBM-MSCs and blue indicates the nuclei. Confocal images were captured as a 6.25 mm2 square at random and the DiI-positive cells were counted manually (N > 20). B Results represent the mean ± standard deviation of two independent experiments
Fig. 5
Fig. 5 Schemes explaining the mechanism how chemerin chemo-attracts the circulating MSCs to the injured liver. Chemerin secreted from the injured liver interacts with ChemR23 on surface of MSCs, leading to activation of adhesion molecules that mediate steps of trans-endothelial migration of the circulating MSCs to the injured liver

그림 출처: PubMed Central 공개 논문 (PMC8252297)

Conflict of interest statement

The authors declare that no competing interests exist.

그 밖의 정보
출판 형태
Journal Article
저널
Cell & bioscience · ISSN 2045-3701
전자 출판
2021-07-01
연구비
Ministry of Health and Welfare HI14C1277
참고문헌
31편