Analysis of differentially expressed genes during the differentiation of human induced pluripotent stem cells and embryonic stem cells into pericytes and endothelial cells_Chinese Journal of Ocular Fundus Diseases

Authors：

Li Jizhu , Ma Yuan , Liu Baoyi , Liu Yaping , Chen Ziye ,  Li Tao

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou 510060, China;

Corresponding author：

Li Tao, Email: litao2@mail.sysu.edu.cn

Keywords：

Human induced pluripotent stem cells; Human embryonic stem cells; Pericytes; Endothelial cells; Transcriptome sequencing

DOI：

10.3760/cma.j.cn511434-20241011-00379

Video：

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[Abstract]Objective To study the differentially expressed genes (DEG) during the differentiation of human induced pluripotent stem cells (hiPSC) and human embryonic stem cells (hESC) into pericytes and endothelial cells, and to identify key molecules and signaling pathways that may regulate this differentiation process. Methods hiPSC and hESC were selected and expanded using mTeSR medium. A "two-step method" was used to induce the differentiation of hiPSC and hESC into pericytes and endothelial cells. Pericytes were identified using immunofluorescence staining, while endothelial cells were isolated and identified using flow cytometry. Total RNA samples were extracted on days 0, 4, 7, and 10 of differentiation and consistently significant DEGs were screened. Gene annotation (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis were performed on the screened DEGs. Results Both hiPSCs and hESCs successfully differentiated into pericytes and endothelial cells under induction conditions. Transcriptome sequencing results showed that with the extension of differentiation time, the DEGs in hiPSCs and hESCs were significantly upregulated or downregulated, following a generally consistent trend. During the differentiation process, marker genes for pericytes and endothelial cells were significantly upregulated. A total of 491 persistent DEGs were detected in both hiPSC and hESC, with 164 unique to hiPSCs and 335 to hESCs, while 8 DEGs were co-expressed in both cell lines. Among these, SLC30A3, LCK, TNFRSF8, PRDM14, and GLB1L3 showed sustained downregulation, whereas CLEC18C, CLEC18B, and F2RL2 exhibited sustained upregulation. GO enrichment analysis revealed that DEGs with sustained upregulation were primarily enriched in terms related to neurogenesis, differentiation, and developmental proteins, while DEGs with sustained downregulation were enriched in terms related to membrane structure and phospholipid metabolic processes. KEGG pathway analysis showed that upregulated genes were primarily enriched in cancer-related pathways, pluripotency regulatory pathways, the Wnt signaling pathway, and the Hippo signaling pathway, whereas downregulated genes were predominantly enriched in metabolism-related pathways. Conclusions During the differentiation of hiPSC and hESC into pericytes and endothelial cells, 8 DEGs exhibit sustained specific expression changes. These changes may promote pericyte and endothelial cell differentiation by activating the Wnt and Hippo pathways, inhibiting metabolic pathways, releasing the maintenance of stem cell pluripotency, affecting the cell cycle, and inhibiting cell proliferation.

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1. Campochiaro PA. Molecular pathogenesis of retinal and choroidal vascular diseases[J]. Prog Retin Eye Res, 2015, 49: 67-81. DOI: 10.1016/j.preteyeres.2015.06.002.
2. Antonetti DA, Klein R, Gardner TW. Diabetic retinopathy[J]. N Engl J Med, 2012, 366(13): 1227-1239. DOI: 10.1056/nejmra1005073.
3. Gamm DM, Wright LS, Capowski EE, et al. Stem cell therapies and retinal disease: toward the next generation[J]. Invest Ophthalmol Vis Sci, 2018, 59(4): 87-96. DOI: 10.1167/iovs.18-23566.
4. 马映雪, 何广辉, 高翔, 等. 人脐带间充质干细胞外泌体靶向miR-126调节高糖诱导的人视网膜血管内皮细胞中血管内皮生长因子-A的表达[J]. 中华眼底病杂志, 2024, 40(5): 372-378. DOI: 10.3760/cma.j.cn511434-20240108-00007.Ma YX, He GH, Gao X, et al. Human umbilical cord mesenchymal stem cell exosomes target miR-126 regulate the expression of vascular endothelial growth factor-A in high glucose-induced human retinal vascular endothelial cells[J]. Chin J Ocul Fundus Dis, 2024, 40(5): 372-378. DOI: 10.3760/cma.j.cn511434-20240108-00007.
5. Medina RJ, O’Neill CL, Humphreys MW, et al. Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy[J]. Invest Ophthalmol Vis Sci, 2010, 51(11): 5906-5913. DOI: 10.1167/iovs.09-4951.
6. Wimmer RA, Leopoldi A, Aichinger M, et al. Human blood vessel organoids as a model of diabetic vasculopathy[J]. Nature, 2019, 565(7740): 505-510. DOI: 10.1038/s41586-018-0858-8.
7. Orlova VV, Drabsch Y, Freund C, et al. Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts[J]. Arterioscler Thromb Vasc Biol, 2014, 34(1): 177-186. DOI: 10.1161/atvbaha.113.302598.
8. Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks[J]. Nat Protoc, 7(3): 562-758. DOI: 10.1038/nprot.2012.016.
9. Huang YL, Pai FS, Tsou YT, et al. Human CLEC18 gene cluster contains C-type lectins with differential glycan-binding specificity[J]. J Biol Chem, 2015, 290(35): 21252-21263. DOI: 10.1074/jbc.m115.649814.
10. Guo RM, Zhao CB, Li P, et al. Overexpression of CLEC18B associates with the proliferation, migration, and prognosis of glioblastoma[J/OL]. ASN Neuro, 2018, 10: 1759091418781949[2018-01-01]. https://pubmed.ncbi.nlm.nih.gov/29914265/. DOI: 10.1177/1759091418781949.
11. Huang SN, Li GS, Zhou XG, et al. Identification of an immune score-based gene panel with prognostic power for oral squamous cell carcinoma[J/OL]. Med Sci Monit, 2020, 26: e922854[2020-06-12]. https://pubmed.ncbi.nlm.nih.gov/32529991/. DOI: 10.12659/MSM.922854.
12. Takebe N, Harris PJ, Warren RQ, et al. Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways[J]. Nat Rev Clin Oncol, 2011, 8(2): 97-106. DOI: 10.1038/nrclinonc.2010.196.
13. Yang L, Shi P, Zhao G, et al. Targeting cancer stem cell pathways for cancer therapy[J]. Signal Transduct Target Ther, 2020, 5(1): 8. DOI: 10.1038/s41392-020-0110-5.
14. Cohen ED, Tian Y, Morrisey EE. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis, and progenitor self-renewal[J]. Development, 2008, 135(5): 789-798. DOI: 10.1242/dev.016865.
15. Nusse R, Clevers H. Wnt/β-Catenin signaling, disease, and emerging therapeutic modalities[J]. Cell, 2017, 169(6): 985-999. DOI: 10.1016/j.cell.2017.05.016.
16. Orlova VV, Drabsch Y, Freund C, et al. Wnt signaling promotes differentiation of human pluripotent stem cells to mesoderm and endoderm lineages while inhibiting neural differentiation[J]. Stem Cells, 2014, 32(10): 2651-2662. DOI: 10.1002/stem.1787.
17. Paik DT, Tian L, Lee J, et al. Large-scale single-cell transcriptomics reveals Wnt-mediated endothelial cell transformation during aortic dissection in mice[J]. J Clin Invest, 2020, 130(9): 4414-4428. DOI: 10.1172/JCI137582.
18. Ma S, Meng Z, Chen R, et al. The Hippo pathway: biology and pathophysiology[J]. Annu Rev Biochem, 2019, 88: 577-604. DOI: 10.1146/annurev-biochem-013118-111829.
19. Zhou Q, Li L, Zhao B, et al. The Hippo pathway in heart development, regeneration, and diseases[J]. Circ Res, 2015, 116(8): 1431-1447. DOI: 10.1161/CIRCRESAHA.116.303218.
20. Choi HJ, Zhang H, Park H, et al. Yes-associated protein regulates endothelial cell contact-mediated expression of angiopoietin-2[J/OL]. Nat Commun, 2015, 6: 6943[2015-05-12]. https://pubmed.ncbi.nlm.nih.gov/25962877/. DOI: 10.1038/ncomms7943.
21. Aguilar JS, Hidalgo A, Lozano J, et al. Directed cardiomyogenesis of human pluripotent stem cells by modulating Wnt/β-catenin and BMP signalling with small molecules[J]. Biochem J, 2015, 469(2): 235-241. DOI: 10.1042/BJ20150245.
22. Parsons SJ, Parsons JT. Src family kinases, key regulators of signal transduction[J]. Oncogene, 2004, 23(48): 7906-7909. DOI: 10.1038/sj.onc.1208160.
23. Boggon TJ, Eck MJ. Structure and regulation of Src family kinases[J]. Oncogene, 2004, 23(48): 7918-7927. DOI: 10.1038/sj.onc.1208081.
24. Meyn MA 3rd, Schreiner SJ, Dumitrescu TP, et al. SRC family kinase activity is required for murine embryonic stem cell growth and differentiation[J]. Mol Pharmacol, 2005, 68(5): 1320-1330. DOI: 10.1124/mol.104.010231.
25. Geijsen N. Epigenetic reprogramming: Prdm14 hits the accelerator[J]. EMBO J, 2012, 31(10): 2247-2248. DOI: 10.1038/emboj.2012.117.
26. Sybirna A, Tang WWC, Pierson Smela M, et al. A critical role of PRDM14 in human primordial germ cell fate revealed by inducible degrons[J/OL]. Nat Commun, 2020, 11(1): 1282[2020-03-09]. https://pubmed.ncbi.nlm.nih.gov/32152282/. DOI: 10.1038/s41467-020-15042-0.
27. Ghosh A, Som A. RNA-Seq analysis reveals pluripotency-associated genes and their interaction networks in human embryonic stem cells[J/OL]. Comput Biol Chem, 2020, 85: 107239[2020-02-21]. https://pubmed.ncbi.nlm.nih.gov/32109853/. DOI: 10.1016/j.compbiolchem.2020.107239.
28. Atsaves V, Lekakis L, Drakos E, et al. The oncogenic JUNB/CD30 axis contributes to cell cycle deregulation in ALK+ anaplastic large cell lymphoma[J]. Br J Haematol, 2014, 167(4): 514-523. DOI: 10.1111/bjh.13079.
29. Lu Y, Wan Z, Zhang X, et al. PRDM14 inhibits 293T cell proliferation by influencing the G1/S phase transition[J]. Gene, 2016, 595(2): 180-186. DOI: 10.1016/j.gene.2016.09.039.
30. Bafaro E, Liu Y, Xu Y, et al. The emerging role of zinc transporters in cellular homeostasis and cancer[J/OL]. Signal Transduct Target Ther, 2017, 2: 17029[2017-07-28]. https://pubmed.ncbi.nlm.nih.gov/29218234/. DOI: 10.1038/sigtrans.2017.29.
31. Kong BY, Duncan FE, Que EL, et al. The inorganic anatomy of the mammalian preimplantation embryo and the requirement of zinc during the first mitotic divisions[J]. Dev Dyn, 2015, 244(8): 935-947. DOI: 10.1002/dvdy.24285.
32. Suetsugu S, Kurisu S, Takenawa T. Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins[J]. Physiol Rev, 2014, 94(4): 1219-1248. DOI: 10.1152/physrev.00040.2013.
33. Ito K, Ito K. Metabolism and the control of cell fate decisions and stem cell renewal[J]. Annu Rev Cell Dev Biol, 2016, 32: 399-409. DOI: 10.1146/annurev-cellbio-111315-125134.

Chinese Journal of Ocular Fundus Diseases

Latest ArticlesAnalysis of differentially expressed genes during the differentiation of human induced pluripotent stem cells and embryonic stem cells into pericytes and endothelial cells

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