Tissue-engineered cartilage regeneration based on human induced pluripotent stem cells

doi:10.12439/kqhm.1005-4979.2024.01.003

Abstract

Abstract:

Objective: To establish a stable and efficient culture approach for guiding human induced pluripotent stem cells (hiPSCs) differentiation towards chondrogenic mesodermal lineage, and preliminarily explore the possibility of hiPSCs-derived cartilaginous pellets for articular cartilage regeneration. Methods: By simulating the development process in vivo, hiPSCs were gradually induced to differentiate into chondrogenic mesodermal lineage and mature cartilage using three dimensions (3D) suspension culture system. Real-time quantitative polymerase chain reaction(RT-qPCR), Western blotting, immunofluorescence and immunohistochemistry were used to investigate the dynamic expression change of pluripotent, mesodermal and chondrongenic related genes and proteins during the whole culture procedure. Finally, subcutaneous model of nude mice was used to investigate the stability of chondrogenic phenotypes and tumorigenicity of hiPSCs derived-cartilaginous pellets.Results: By simulating the development process in vivo, hiPSCs were gradually induced to differentiate into chondrogenic mesodermal lineage, and finally differentiated into mature cartilage tissues. Conclusion: In this study, a stable and efficient culture approach for inducing hiPSCs differentiation towards chondrogenic mesoderm was successfully established, which provide a research platform for investigation of hiPSCs-mediated cartilage development and regeneration.

Key words: human induced pluripotent stem cells, mesoderm, suspension culture, cartilaginous pellets

摘要：

目的：构建一种稳定高效的人诱导多能干细胞（induced pluripotent stem cells，iPSCs）向软骨中胚层方向分化的培养方法，初步探究hiPSCs来源的软骨用于关节软骨再生的可能。方法：通过模拟体内发育过程和三维（three dimensions，3D）悬浮培养体系，逐步诱导hiPSCs向软骨中胚层和成熟的软骨组织分化；利用实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）、蛋白质印迹法（Western blotting）、免疫荧光及免疫组织化学技术检测整个培养过程中多能干性、中胚层及软骨相关基因和蛋白的动态变化；最后利用裸鼠皮下模型研究hiPSCs软骨球软骨表型的稳定性及成瘤性。结果：通过模拟体内发育过程，成功逐步诱导hiPSCs向软骨中胚层方向分化，最终分化为成熟的软骨组织。结论：本研究成功构建了一种稳定高效诱导hiPSCs向软骨中胚层方向分化的培养体系，为探究hiPSCs介导的软骨组织发育和再生提供了一个研究平台。

关键词: 人类诱导多能干细胞, 中胚层, 悬浮培养, 软骨球

CLC Number:

R782

LI Yangyang, ZHANG Maolin, ZOU Duohong, ZHANG Zhiyuan. Tissue-engineered cartilage regeneration based on human induced pluripotent stem cells[J]. 《Journal of Oral and Maxillofacial Surgery》, 2024, 34(1): 22-28.

李阳阳, 张茂林, 邹多宏, 张志愿. 基于人诱导多能干细胞的组织工程化软骨再生[J]. 《口腔颌面外科杂志》, 2024, 34(1): 22-28.

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URL: https://journal06.magtech.org.cn/Jweb_joms/EN/10.12439/kqhm.1005-4979.2024.01.003

https://journal06.magtech.org.cn/Jweb_joms/EN/Y2024/V34/I1/22

Figures/Tables 5

Table 1 Primers sequence for RT-qPCR

基因	正向引物(5'-3')	反向引物(5'-3')
ACTB	TGGCACCACACCTTCTACAATGAGC	GCACAGCTTCTCCTTAATGTCACGC
OCT3/4	GACAACAATGAGAACCTTCA	TTCTGGCGCCGGTTACAGAA
Nang	CAAAGGCAAACAACCCACTT	CTGGATGTTCTGGGTCTGGT
Brachyury	TATGAGCCTCGAATCCACATAGT	CCTCGTTCTGATAAGCAGTCAC
Sox9	AGCGAACGCACATCAAGAC	CTGTAGGCGATCTGTTGGGG
Col2a1	TTTCCCAGGTCAAGATGGTC	CTTCAGCACCTGTCTCACCA

Figure 1 In vitro culture and pluripotency maintenance of hiPSCs（×100）

Figure 2 Culture system of hiPSCs cartilage mesodermal induced differentiation

Figure 3 Expression analysis of markers Nanog, Brachyury, Sox9 and Col2a1, and histological analysis of hiPSCs-derived cartilaginous pellets

Figure 4 Subcutaneous transplantation of hiPSCs-derived cartilaginous pellets and histological analysis (×100)

References 23

[1]	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J]. Cell, 2006, 126(4): 663-676. doi: 10.1016/j.cell.2006.07.024 pmid: 16904174
[2]	Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: Immune evasive, not immune privileged[J]. Nat Biotechnol, 2014, 32(3): 252-260. doi: 10.1038/nbt.2816 pmid: 24561556
[3]	Kristiansen CK, Chen AB, Høyland LE, et al. Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations[J]. Cell Cycle, 2022, 21(20): 2206-2221. doi: 10.1080/15384101.2022.2092185 URL
[4]	Mungenast AE, Siegert S, Tsai LH. Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells[J]. Mol Cell Neurosci, 2016, 73: 13-31. doi: 10.1016/j.mcn.2015.11.010 pmid: 26657644
[5]	Aboul-Soud MAM, Alzahrani AJ, Mahmoud A. Induced pluripotent stem cells (iPSCs)-roles in regenerative therapies, disease modelling and drug screening[J]. Cells, 2021, 10(9): 2319. doi: 10.3390/cells10092319 URL
[6]	Mulfaul K, Giacalone JC, Voigt AP, et al. Stepwise differentiation and functional characterization of human induced pluripotent stem cell-derived choroidal endothelial cells[J]. Stem Cell Res Ther, 2020, 11(1): 409. doi: 10.1186/s13287-020-01903-4 pmid: 32967716
[7]	Dewell TE, Gjoni K, Liu AZ, et al. Transcription factor overexpression drives reliable differentiation of retinal pigment epithelium from human induced pluripotent stem cells[J]. Stem Cell Res, 2021, 53: 102368. doi: 10.1016/j.scr.2021.102368 URL
[8]	Cao H, Zhou Q, Liu CG, et al. Substrate stiffness regulates differentiation of induced pluripotent stem cells into heart valve endothelial cells[J]. Acta Biomater, 2022, 143: 115-126. doi: 10.1016/j.actbio.2022.02.032 pmid: 35235867
[9]	Netsrithong R, Suwanpitak S, Boonkaew B, et al. Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells[J]. Stem Cell Res Ther, 2020, 11(1): 481. doi: 10.1186/s13287-020-01997-w pmid: 33176890
[10]	Liu LP, Li YM, Guo NN, et al. Therapeutic potential of patient iPSC-derived iMelanocytes in autologous transplantation[J]. Cell Rep, 2019, 27(2): 455-466.e5. doi: S2211-1247(19)30362-6 pmid: 30970249
[11]	Ramzy A, Thompson DM, Ward-Hartstonge KA, et al. Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes[J]. Cell Stem Cell, 2021, 28(12): 2047-2061.e5. doi: 10.1016/j.stem.2021.10.003 pmid: 34861146
[12]	Dicks AR, Steward N, Guilak F, et al. Chondrogenic differentiation of human-induced pluripotent stem cells[J]. Methods Mol Biol, 2023, 2598: 87-114. doi: 10.1007/978-1-0716-2839-3_8 pmid: 36355287
[13]	Wei Y, Zeng W, Wan R, et al. Chondrogenic differentiation of induced pluripotent stem cells from osteoarthritic chondrocytes in alginate matrix[J]. Eur Cell Mater, 2012, 23: 1-12. pmid: 22241609
[14]	Hu YQ, Chen L, Gao Y, et al. A lithium-containing biomaterial promotes chondrogenic differentiation of induced pluripotent stem cells with reducing hypertrophy[J]. Stem Cell Res Ther, 2020, 11(1): 77. doi: 10.1186/s13287-020-01606-w pmid: 32085810
[15]	Zhang ML, Shi JF, Xie M, et al. Recapitulation of cartilage/bone formation using iPSCs via biomimetic 3D rotary culture approach for developmental engineering[J]. Biomaterials, 2020, 260: 120334. doi: 10.1016/j.biomaterials.2020.120334 URL
[16]	Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J]. Cell, 2007, 131(5): 861-872. doi: 10.1016/j.cell.2007.11.019 pmid: 18035408
[17]	Orozco-Fuentes S, Neganova I, Wadkin LE, et al. Quantification of the morphological characteristics of hESC colonies[J]. Sci Rep, 2019, 9(1): 17569. doi: 10.1038/s41598-019-53719-9 pmid: 31772193
[18]	Kobayashi T, Surani MA. On the origin of the human germline[J]. Development, 2018, 145(16): dev150433. doi: 10.1242/dev.150433 URL
[19]	Abramoff B, Caldera FE. Osteoarthritis: Pathology, diagnosis, and treatment options[J]. Med Clin North Am, 2020, 104(2): 293-311. doi: 10.1016/j.mcna.2019.10.007 URL
[20]	Edmondson R, Broglie JJ, Adcock AF, et al. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors[J]. ASSAY Drug Dev Technol, 2014, 12(4): 207-218. doi: 10.1089/adt.2014.573 pmid: 24831787
[21]	Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art[J]. Tissue Eng Part B Rev, 2008, 14(1): 61-86. doi: 10.1089/teb.2007.0150 URL
[22]	Abousleiman RI, Reyes Y, McFetridge P, et al. Tendon tissue engineering using cell-seeded umbilical veins cultured in a mechanical Stimulator[J]. Tissue Eng Part A, 2009, 15(4): 787-795. doi: 10.1089/ten.tea.2008.0102 URL
[23]	Yamashita A, Morioka M, Yahara Y, et al. Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs[J]. Stem Cell Reports, 2015, 4(3): 404-418. doi: 10.1016/j.stemcr.2015.01.016 pmid: 25733017

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