Exosomes derived from feline adipose tissue mesenchymal stem cells reduce inflammation factors Soo-Eun Sung , Kyung-Ku Kang , Joo-Hee Choi, Si-Joon Lee , MinKyoung Sung , Kil-Soo Kim, Min-Soo Seo* 1 Laboratory animal center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea Abstract Results Adipose tissue derived mesenchymal stem cells (AD-MSCs) release extracellular vesicles such as exosomes and microparticles. Particularly, exosomes are formed inward a cell, throughout multi- vesicular bodies (MVB), so the contents protein, DNA, and RNA are similar to the parent
cells. The exosomes research is rapidly expanding, many studies published in recent years, but the function Figure 1. Characterization and application of MSC-derived exosomes should emerge as cell- of feline AD-MSCs. free therapeutics compared with well-known mesenchymal stem cells (A) Isolated feline therapeutic effects. We isolated exosomes using ultracentrifugation fibroblasts. (B) feline AD- from feline AD-MSCs and feline fibroblasts cell culture medium. MSC morphology Characterization of feline exosomes using FACS, DLS, NTA and throughout microscopy TEM imaging. The exosomes had typical
size about 100 nm and (original magnification 100×) expressed tetraspanins such as CD9, CD81. And then, we analyzed (C) Feline AD-MSC cytokines and chemokines levels from exosomes by ELISA. As a analyzed stem cells positive result, anti-inflammatory factor IL-10 increased in AD-MSCs (CD105, CD90, and CD44) exosomes, pro-inflammatory factors such as IL-1 beta, IL-8 and IFN- and negative (CD14, CD34 gamma decreased in AD-MSC exosomes. Our work is the first and CD45) markers by flow demonstrated that feline AD-MSCs derived exosomes enhanced the cytometry. inflammatory suppressive effects and it
had the therapeutic potential of immune disease. Keywords Mesenchymal stem cells, AD-MSCs, Exosomes, Extracellular vesicles, Anti-inflammation. Figure 2. Characterization Introduction of feline AD-MSC and fibroblasts exosomes. Application of mesenchymal stem cells with multiple lineage for (A) Electron micrographs of regeneration of various tissue damage such as skin, cartilage, bone, isolated exosomes with the adipose, and muscle and so on. A large number of experimental typical morphology and size. studies on the tissue regenerative medicine that MSCs secrete many (B) Exosome marker factors
to modulate angiogenesis for regeneration or anti- detection by flow cytometry. inflammation [1]. Extracellular vesicles (EVs) including exosomes, (C) NTA analysis of microparticles, membrane particles and apoptotic vesicles. They are exosome particles number released from various cells under normal of pathological conditions and size distribution. so they are contain genetic information [2]. Exosome isolation is possible many biofluids such as serum, plasma, cerebrospinal fluid (CSF), urine and saliva not only cell culture supernatants [3]. In this work, we isolated exosomes from cell culture
medium to compare Figure 3. Comparing feline AD-MSCs and dermal fibroblasts derived exosomes. cytokine and chemokine levels Methods between feline fibroblasts derived [Cell isolation] [Exosome isolation] exosomes and feline AD-MSCs derived exosomes. (Y-axis indicates fluorescence intensity (a.u), *p-value < 0.05, **≤0.0001) Conclusions In conclusion, isolated feline AD-MSCs derived exosomes reduced inflammation effects throughout low expression of pro-inflammatory cytokines and chemokine secretion and high level of anti- inflammation factor IL-10. Feline AD-MSC and human AD-MSCs display
similar proliferative capacity and immune-suppressive functions. So these data will support that exosomes have References immunotherapeutic potentials then it can be used for medicine related to inflammation diseases. [1] Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J: Mesenchymal Stem Cells for Regenerative Medicine. Cells 2019, 8(8). [2] van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R: Acknowledgements Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 2012, 64(3):676-705. We would like to thank laboratory animal resources bank (LAREB) for [3]
Bucan V, Vaslaitis D, Peck CT, Strauss S, Vogt PM, Radtke C: transfer the feline AD-MSCs. This project was by a grant of NLAR Effect of Exosomes from Rat Adipose-Derived Mesenchymal (National Laboratory Animal Resources) from the Ministry of Food Stem Cells on Neurite Outgrowth and Sciatic Nerve Regeneration and Drug Safety in 2019-2021. After Crush Injury. Mol Neurobiol 2019, 56(3):1812-1824. [X. Stem cell biology-1] Exosomes derived from feline adipose tissue mesenchymal stem cells reduce inflammation factors Soo-Eun Sung¹, Kyung-Ku Kang¹, Joo-Hee Choi¹, Si-Joon Lee¹, Minkyoung Sung¹,
Kil-Soo Kim¹, Min-Soo Seo¹˙* ¹Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea Adipose tissue derived mesenchymal stem cells (AD-MSCs) release extracellular vesicles such as exosomes and microparticles. Particularly, exosomes are formed inward a cell, throughout multivesicular bodies (MVB), so the contents protein, DNA, and RNA are similar to the parent cells. The exosomes research is rapidly expanding, many studies published in recent years, but the function and application of MSC-derived exosomes should emerge as cell-free therapeutics
compared with well-known mesenchymal stem cells therapeutic effects. We isolated exosomes using ultracentrifugation from feline AD-MSCs and feline fibroblasts cell culture medium. Characterization of feline exosomes using FACS, DLS, NTA and TEM imaging. The exosomes had typical size about 100 nm and expressed tetraspanins such as CD9, CD81. And then, we analyzed cytokines and chemokines levels from exosomes by ELISA. As a result, anti-inflammatory factor IL-10 increased in AD-MSCs exosomes, pro-inflammatory factors such as IL-1 beta, IL-8 and IFN-gamma decreased in AD-MSC exosomes. Our work is
the first demonstrated that feline AD- MSCs derived exosomes enhanced the inflammatory suppressive effects and it had the therapeutic potential of immune disease. Safe Scarless Cassette-free Selection of Genome-edited Human Pluripotent Stem Cells Using Temporary Drug Resistance 3,4 5 2 Keun-Tae Kim *, Ju-Chan Park *, Hyeon-Ki Jang *, Haeseung Lee , Seokwoo Park , Jumee Kim , Ok-Seon Kwon , Young-Hyun Go , Yan Jin , Wankyu Kim , 1 1 2 5 7 2 6 Jeongmi Lee , Sangsu Bae 4 and Hyuk-Jin Cha 2# 3, 7 1Department of Life Sciences, Sogang University, Seoul, Republic of Korea, 2College of Pharmacy, Seoul
National University, Seoul, Republic of Korea, 3Department of Chemistry, Hanyang University, Seoul, Republic of Korea, 4Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea, 5Ewha Research Center for Systems Biology, Division of Molecular & Life Sciences, Ewha Womans University, Seoul, Republic of Korea, 6Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea, 7School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Republic of Korea Abstract An efficient gene editing technique for use in human
pluripotent stem cells (hPSCs) would have great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the substantially low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved. Previously, we demonstrated that YM155, a Survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high
expression of SLC35F2 in these cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced, thereby achieving enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes. RESULTS G Figure
1. High expression of SLC35F2 mediates intracellular uptake of YM155 in hPSCs. (A) A graphical scheme of correlating cell line hPSC scores with cell line drug responses (AUC) Figure 4. YM155 mediated enriched selection in HEK293T. (A) Relative against each of 543 compounds available in CTRP (B) Correlation of 543 compounds from comparing expression of SLC35F2 in several cells including HEK293T cells through NextBio portal. (B) Graphical scheme of YM155 mediated enriched selection (YES) approach in HEK293T cells. (C) AUCs of each compound to hPSC (left) scores and YM155 sensitivity (right). (C)
Expression of SLC35F2 in Scheme of CCR5 surrogate vector system. (D) Quantified amount of GFP+ population after several hESCs from NextBio portal. (D, E) SLC35F2 and POU5F1 gene expression in differentiated cells YES-approach in HEK293T cells. (E) T7E1 assay of samples from (D) (TS: Surrogate vector only, (hDF: human dermal fibroblast) and hESC (CHA3), hiPSC(SES8) cells. (F) IFC of ɣH2AX in hESCs after C: CCR5 sgRNA only, S: SLC35F2 sgRNA only, C+S: CCR5 and SLC35F2 sgRNA transfected). YM155 treatment. (G) LC-MS/MS analysis for intracellular YM155 amount between hDF and hESC. (H) (F)
Fluorescence microscopic images of from (D). (G) Graph indicates the deep sequencing analysis targeting CCR5 after YES approach in HEK293T cells. (H) Cell viability of SLC35F2 Graphical scheme of selective uptake of YM155 in hPSCs. reconstituted KO cells after YM155 treatment. Figure 7. Scarless YES-approach for multiple knockout targets and knock-in models. (A) T7E1 analysis and (B) indel frequencies with and without YES-approach in CCR5, HEK2 and HEK3 targets. (C) Fluorescent images of Cas9-EGFP #1 hESCs (left) and graphical diagram for determining target efficiency through EGFP reporter
system (right). (D) Flow cytometry of EGFP+ and EGFP- population after YES approach. (E, F) Graphical diagram of knock-in targets at EYA4, TMEM67 and SLC6A5 loci. (F) Target efficiency of KO and KI of indicative genes. (G) Plasmid donor DNA-based large insertion targeting AAVS locus with EYFP in hESCs. B Figure 2. SLC35F2 enables YM155-induced selective cell death in hPSCs. (A) Graphical scheme of SLC35F2 target sequence. (B) Experimental scheme of SLC35F2 KO enrichment (top) and Annexin-V assay after YM155 treatment (bottom). (C) T7E1 assay after YM155 treatment in SLC35F2 sgRNA transfected
hESCs. (D) Sequence information of SLC35F2 KO single clone (KO#1). (E) Annexin-V assay and (F) IFC for ɣH2AX of KO#1 cells after YM155 treatment. (G) LC-MS/MS analysis for intracellular YM155 amount in NC and SLC35F2 KO #1 hESCs after YM155 treatment. (H) Phase contrast image of SLC35F2 KO#5 hESCs after YM155 treatment. (I) SLC35F2 gene expression of KO#1 and KO#5 Figure 5. YM155 based enriched selection of genome-edited hESCs. (A) hESCs through RT-qPCR (top) and RT-PCR (bottom) (RNA18S for loading control). Graphical scheme of YES-approach with indicative raio of sgRNAs in hESCs. (C: CCR5, S:
SLC35F2 sgRNA). (B) T7E1 assay for CCR5 and SLC35F2 in each clone of hESCs. (C) Graphical presentation of Indel mutation by indicative conditions (left) and list of read counts based on deep sequencing (right table). Figure 8. Off-target effect of scarless YES approach Off-target effects were validated on multiple targets using scarless YES approach. Putative off-target sites were predicted through off-finder CONCLUSION Figure 6. Scarless YES-approach for establishment of CCR5 targeted hESCs. (A) Graphical scheme of scarless YES-approach. (B) Relative SLC35F2 gene expression followed by days
after siSLC35F2 transfection in hESC
