Abstract
The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.
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References
Mansour JM (2003) Biomechanics of Cartilage. In: Kinesiology: the mechanics and pathomechanics of human movement, vol 2e, pp 66–75
Guilak F (2011) Biomechanical factors in osteoarthritis. Best Pract Res Clin Rheumatol 25(6):815–823. https://doi.org/10.1016/j.berh.2011.11.013
Antons J, Marascio MGM, Nohava J et al (2018) Zone-dependent mechanical properties of human articular cartilage obtained by indentation measurements. J Mater Sci Mater Med 29(5):57. https://doi.org/10.1007/s10856-018-6066-0
Sophia Fox AJ, Bedi A, Rodeo SA (2009) The basic science of articular cartilage: structure, composition, and function. Sports Health 1(6):461–468. https://doi.org/10.1177/1941738109350438
Xia Y, Momot KI, Chen Z et al (2017) Chapter 1 Introduction to Cartilage. In: Biophysics and biochemistry of Cartilage by NMR and MRI. The Royal Society of Chemistry, pp 1–43. https://doi.org/10.1039/9781782623663-00001
Zhang L, Hu J, Athanasiou KA (2009) The role of tissue engineering in articular cartilage repair and regeneration. Crit Rev Biomed Eng 37(1–2):1–57. https://doi.org/10.1615/critrevbiomedeng.v37.i1-2.10
Guilak F, Hung C (2005) Physical regulation of cartilage metabolism. In: Mow V, Huiskes R (eds) Basic orthopaedic biomechanics and mechanobiology, 3rd edn. Lippincott-Raven Publishers, Philadelphia, pp 179–207
Shieh AC, Athanasiou KA (2003) Principles of cell mechanics for cartilage tissue engineering. Ann Biomed Eng 31(1):1–11. https://doi.org/10.1114/1.1535415
Sanchez-Adams J, Leddy HA, McNulty AL et al (2014) The mechanobiology of articular cartilage: bearing the burden of osteoarthritis. Curr Rheumatol Rep 16(10):451. https://doi.org/10.1007/s11926-014-0451-6
Berenbaum F, Griffin TM, Liu-Bryan R (2017) Review: metabolic regulation of inflammation in osteoarthritis. Arthritis Rheumatol 69(1):9–21. https://doi.org/10.1002/art.39842
Breedveld FC (2004) Osteoarthritis--the impact of a serious disease. Rheumatology (Oxford) 43(Suppl 1):i4–i8. https://doi.org/10.1093/rheumatology/keh102
Smolen JS, Aletaha D, McInnes IB (2016) Rheumatoid arthritis. Lancet 388(10055):2023–2038. https://doi.org/10.1016/S0140-6736(16)30173-8
Barbour KE, Moss S, Croft JB et al (2018) Geographic variations in arthritis prevalence, health-related characteristics, and management - United States, 2015. MMWR Surveill Summ 67(4):1–28. https://doi.org/10.15585/mmwr.ss6704a1
Palazzo C, Nguyen C, Lefevre-Colau MM et al (2016) Risk factors and burden of osteoarthritis. Ann Phys Rehabil Med 59(3):134–138. https://doi.org/10.1016/j.rehab.2016.01.006
Willard VP, Diekman BO, Sanchez-Adams J et al (2014) Use of cartilage derived from murine induced pluripotent stem cells for osteoarthritis drug screening. Arthritis Rheumatol 66(11):3062–3072. https://doi.org/10.1002/art.38780
Adkar SS, Brunger JM, Willard VP et al (2017) Genome engineering for personalized arthritis therapeutics. Trends Mol Med 23(10):917–931. https://doi.org/10.1016/j.molmed.2017.08.002
O’Connor SK, Katz DB, Oswald SJ et al (2020) Formation of Osteochondral organoids from murine induced Pluripotent stem cells. Tissue Eng Part A. https://doi.org/10.1089/ten.TEA.2020.0273
Saitta B, Passarini J, Sareen D et al (2014) Patient-derived skeletal dysplasia induced pluripotent stem cells display abnormal chondrogenic marker expression and regulation by BMP2 and TGFbeta1. Stem Cells Dev 23(13):1464–1478. https://doi.org/10.1089/scd.2014.0014
Nur Patria Y, Stenta T, Lilianty J et al (2020) CRISPR/Cas9 gene editing of a SOX9 reporter human iPSC line to produce two TRPV4 patient heterozygous missense mutant iPSC lines, MCRIi001-A-3 (TRPV4 p.F273L) and MCRIi001-A-4 (TRPV4 p.P799L). Stem Cell Res 48:101942. https://doi.org/10.1016/j.scr.2020.101942
Liu H, Yang L, Yu FF et al (2017) The potential of induced pluripotent stem cells as a tool to study skeletal dysplasias and cartilage-related pathologic conditions. Osteoarthr Cartil 25(5):616–624. https://doi.org/10.1016/j.joca.2016.11.015
Sanjurjo-Rodriguez C, Castro-Vinuelas R, Pineiro-Ramil M et al (2020) Versatility of induced Pluripotent stem cells (iPSCs) for improving the knowledge on musculoskeletal diseases. Int J Mol Sci 21(17). https://doi.org/10.3390/ijms21176124
Yamashita A, Morioka M, Kishi H et al (2014) Statin treatment rescues FGFR3 skeletal dysplasia phenotypes. Nature 513(7519):507–511. https://doi.org/10.1038/nature13775
Matsumoto Y, Hayashi Y, Schlieve CR et al (2013) Induced pluripotent stem cells from patients with human fibrodysplasia ossificans progressiva show increased mineralization and cartilage formation. Orphanet J Rare Dis 8:190. https://doi.org/10.1186/1750-1172-8-190
Miller EE, Kobayashi GS, Musso CM et al (2017) EIF4A3 deficient human iPSCs and mouse models demonstrate neural crest defects that underlie Richieri-Costa-Pereira syndrome. Hum Mol Genet 26(12):2177–2191. https://doi.org/10.1093/hmg/ddx078
Okada M, Ikegawa S, Morioka M et al (2015) Modeling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cells. Hum Mol Genet 24(2):299–313. https://doi.org/10.1093/hmg/ddu444
Loh KM, Chen A, Koh PW et al (2016) Mapping the pairwise choices leading from Pluripotency to human bone, heart, and other mesoderm cell types. Cell 166(2):451–467. https://doi.org/10.1016/j.cell.2016.06.011
Umeda K, Zhao J, Simmons P et al (2012) Human chondrogenic paraxial mesoderm, directed specification and prospective isolation from pluripotent stem cells. Sci Rep 2:455. https://doi.org/10.1038/srep00455
Oldershaw RA, Baxter MA, Lowe ET et al (2010) Directed differentiation of human embryonic stem cells toward chondrocytes. Nat Biotechnol 28(11):1187–1194. https://doi.org/10.1038/nbt.1683
Diekman BO, Christoforou N, Willard VP et al (2012) Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells. Proc Natl Acad Sci U S A 109(47):19172–19177. https://doi.org/10.1073/pnas.1210422109
Adkar SS, Wu CL, Willard VP et al (2019) Step-wise chondrogenesis of human induced Pluripotent stem cells and purification via a reporter Allele generated by CRISPR-Cas9 genome editing. Stem Cells 37(1):65–76. https://doi.org/10.1002/stem.2931
Lim J, Tu X, Choi K et al (2015) BMP-Smad4 signaling is required for precartilaginous mesenchymal condensation independent of Sox9 in the mouse. Dev Biol 400(1):132–138. https://doi.org/10.1016/j.ydbio.2015.01.022
Patil AS, Sable RB, Kothari RM (2011) An update on transforming growth factor-beta (TGF-beta): sources, types, functions and clinical applicability for cartilage/bone healing. J Cell Physiol 226(12):3094–3103. https://doi.org/10.1002/jcp.22698
Tang QO, Shakib K, Heliotis M et al (2009) TGF-beta3: a potential biological therapy for enhancing chondrogenesis. Expert Opin Biol Ther 9(6):689–701. https://doi.org/10.1517/14712590902936823
Craft AM, Rockel JS, Nartiss Y et al (2015) Generation of articular chondrocytes from human pluripotent stem cells. Nat Biotechnol 33(6):638–645. https://doi.org/10.1038/nbt.3210
Yamashita A, Morioka M, Yahara Y et al (2015) Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs. Stem Cell Rep 4(3):404–418. https://doi.org/10.1016/j.stemcr.2015.01.016
Johnstone B, Hering TM, Caplan AI et al (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238(1):265–272. https://doi.org/10.1006/excr.1997.3858
Dicks A, Wu CL, Steward N et al (2020) Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter. Stem Cell Res Ther 11(1):66. https://doi.org/10.1186/s13287-020-01597-8
Wu CL, Dicks A, Steward N et al (2021) Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis. Nat Commun 12(1):362. https://doi.org/10.1038/s41467-020-20598-y
Yamashita A, Tamamura Y, Morioka M et al (2018) Considerations in hiPSC-derived cartilage for articular cartilage repair. Inflamm Regen 38:17. https://doi.org/10.1186/s41232-018-0075-8
Murphy C, Mobasheri A, Tancos Z et al (2018) The potency of induced Pluripotent stem cells in Cartilage regeneration and osteoarthritis treatment. Adv Exp Med Biol 1079:55–68. https://doi.org/10.1007/5584_2017_141
Krishnan Y, Grodzinsky AJ (2018) Cartilage diseases. Matrix Biol 71-72:51–69. https://doi.org/10.1016/j.matbio.2018.05.005
Musunuru K (2013) Genome editing of human pluripotent stem cells to generate human cellular disease models. Dis Model Mech 6(4):896–904. https://doi.org/10.1242/dmm.012054
Yumlu S, Stumm J, Bashir S et al (2017) Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9. Methods 121-122:29–44. https://doi.org/10.1016/j.ymeth.2017.05.009
O’Conor CJ, Case N, Guilak F (2013) Mechanical regulation of chondrogenesis. Stem Cell Res Ther 4(4):61. https://doi.org/10.1186/scrt211
Brunger JM, Zutshi A, Willard VP et al (2017) Genome engineering of stem cells for autonomously regulated, closed-loop delivery of biologic drugs. Stem Cell Rep 8(5):1202–1213. https://doi.org/10.1016/j.stemcr.2017.03.022
Pferdehirt L, Ross AK, Brunger JM et al (2019) A synthetic gene circuit for self-regulating delivery of biologic drugs in engineered tissues. Tissue Eng Part A 25(9–10):809–820. https://doi.org/10.1089/ten.TEA.2019.0027
Nims RJ, Pferdehirt L, Ho NB et al (2021) A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues. Sci Adv 7(5). https://doi.org/10.1126/sciadv.abd9858
Guilak F, Pferdehirt L, Ross AK et al (2019) Designer stem cells: genome engineering and the next generation of cell-based therapies. J Orthop Res 37(6):1287–1293. https://doi.org/10.1002/jor.24304
Tang R, Jing L, Willard VP et al (2018) Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells. Stem Cell Res Ther 9(1):61. https://doi.org/10.1186/s13287-018-0797-1
Thirion S, Berenbaum F (2004) Culture and phenotyping of chondrocytes in primary culture. Methods Mol Med 100:1–14. https://doi.org/10.1385/1-59259-810-2:001
Nasrabadi D, Rezaeiani S, Eslaminejad MB et al (2018) Improved protocol for chondrogenic differentiation of bone marrow derived mesenchymal stem cells -effect of PTHrP and FGF-2 on TGFbeta1/BMP2-induced chondrocytes hypertrophy. Stem Cell Rev Rep 14(5):755–766. https://doi.org/10.1007/s12015-018-9816-y
Estes BT, Diekman BO, Gimble JM et al (2010) Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 5(7):1294–1311. https://doi.org/10.1038/nprot.2010.81
Caplan AI (2017) Mesenchymal stem cells: time to change the name! Stem Cells Transl Med 6(6):1445–1451. https://doi.org/10.1002/sctm.17-0051
Yang YK, Ogando CR, Wang See C et al (2018) Changes in phenotype and differentiation potential of human mesenchymal stem cells aging in vitro. Stem Cell Res Ther 9(1):131. https://doi.org/10.1186/s13287-018-0876-3
Drela K, Stanaszek L, Nowakowski A et al (2019) Experimental strategies of mesenchymal stem cell propagation: adverse events and potential risk of functional changes. Stem Cells Int 2019:7012692. https://doi.org/10.1155/2019/7012692
Takahashi K, Tanabe K, Ohnuki M et al (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872. https://doi.org/10.1016/j.cell.2007.11.019
Nejadnik H, Diecke S, Lenkov OD et al (2015) Improved approach for chondrogenic differentiation of human induced pluripotent stem cells. Stem Cell Rev Rep 11(2):242–253. https://doi.org/10.1007/s12015-014-9581-5
Lee J, Taylor SE, Smeriglio P et al (2015) Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells. FASEB J 29(8):3399–3410. https://doi.org/10.1096/fj.14-269720
Suchorska WM, Augustyniak E, Richter M et al (2017) Comparison of four protocols to generate chondrocyte-like cells from human induced Pluripotent stem cells (hiPSCs). Stem Cell Rev Rep 13(2):299–308. https://doi.org/10.1007/s12015-016-9708-y
Lian Q, Zhang Y, Zhang J et al (2010) Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice. Circulation 121(9):1113–1123. https://doi.org/10.1161/CIRCULATIONAHA.109.898312
Xu M, Shaw G, Murphy M et al (2019) Induced Pluripotent stem cell-derived mesenchymal stromal cells are functionally and genetically different from bone marrow-derived mesenchymal stromal cells. Stem Cells 37(6):754–765. https://doi.org/10.1002/stem.2993
Chen IP, Fukuda K, Fusaki N et al (2013) Induced pluripotent stem cell reprogramming by integration-free Sendai virus vectors from peripheral blood of patients with craniometaphyseal dysplasia. Cell Reprogram 15(6):503–513. https://doi.org/10.1089/cell.2013.0037
Crowe AR, Yue W (2019) Semi-quantitative determination of protein expression using immunohistochemistry staining and analysis: an integrated protocol. Bio Protoc 9(24):10.21769/BioProtoc.3465
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1(3):1559–1582. https://doi.org/10.1038/nprot.2006.236
Acknowledgements
This work was supported by the Shriners Hospitals for Children, Nancy Taylor Foundation, Arthritis Foundation, NIH (AG46927, AG15768, AR075899, AR072999, AR074992, AR073752, AR080902, T32 DK108742, T32 EB018266), and Taiwan GSSA Scholarship.
Conflicts of Interest
FG is an employee of Cytex Therapeutics, Inc. The other authors declare that they have no competing interests.
Author Contribution Statement
ARD, NS, CLW, and FG were involved in the development, testing, and troubleshooting of these protocols and the writing of this paper.
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Dicks, A.R., Steward, N., Guilak, F., Wu, CL. (2023). Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells. In: Stoddart, M.J., Della Bella, E., Armiento, A.R. (eds) Cartilage Tissue Engineering. Methods in Molecular Biology, vol 2598. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2839-3_8
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DOI: https://doi.org/10.1007/978-1-0716-2839-3_8
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