Forward programming human induced pluripotent stem cells into chondrocytes by forced expression of SOX9
Cell Biology
Final Report Abstract
A major problem with chondrocytes derived in vitro from stem cells such as bone marrow-derived mesenchymal stem cells (BMSCs) is undesired hypertrophic degeneration, to which articular chon- drocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes is, however, afflicted with a high cell loss and whether an AC-phenotype and resistance to hypertrophy can be achieved, has not been demonstrated so far. Aim was to engi- neer chondrocytes from human iPSCs that are capable to form non-hypertrophic cartilage and can thus replace ACs in cartilage regenerative medicine and other applications. The primary hypothesis was that overexpression of the chondrogenic master transcription factor SOX9 may overcome currently limited in vitro chondrogenic differentiation of iPSCs and will allow forward programming of hiPSCs into chondrocytes. SOX9 overexpression was established successfully, but iPSC chondrogenesis appeared highly sen- sitive to detrimental effects of lentiviral infection and SOX9 could not rescue negative virus effects at any of the investigated developmental stages. Overexpressing SOX9 in BMSCs at the onset of chondrogenesis was not sufficient for shifting differentiation into the desired chondral instead of the undesired osteochondral lineage. During differentiation of non-transfected iPSCs, limited cell survival correlated with low ex- pression levels of many extracellular matrix-related genes and integrin-signaling pathway-related molecules as well as an inferior aggregation capacity of intermediate mesodermal progenitors at the onset of chondrogenesesis. A short WNT/β-catenin activation pulse at initiation of iPSC dif- ferentiation was a key step to allow the vast majority of the intermediate mesodermal progenitors to participate in pellet formation by stimulating extracellular matrix-related gene expression and strongly improving cell aggregation. This overcame the high cell loss during chondrogenesis. Moreover, we established a novel differentiation protocol for human iPSCs that drives chon- drogenesis with TGF-β without addition of pro-hypertrophic bone morphogenetic proteins. The resulting iPSC-chondrocytes were highly reminiscent of juvenile chondrocytes and deposited more proteoglycans per cell than adult ACs. Importantly, hypertrophic markers in iPSC-cartilage were absent or as low as in AC-tissue both on expression and protein level, thus documenting the absence of undesired hypertrophic degeneration that disqualifies BMSCs for clinical cartilage re- generation. This is the first experimental demonstration of a chondral in vitro differentiation of human iPSCs into chondrocytes resistant to hypertrophic degeneration. Thus, the aim of the project was reached, albeit by an alternate approach. The novel revelations of this project open new strategies for applying stem cells in clinical cartilage regeneration, disease modeling and pharmacological studies.
Publications
- Gegensätzliche frühe Regulation von SOX9-Protein bei der In-vitro-Chondrogenese von iPS-Zellen im Vergleich zu MSCs. Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2016)
Diederichs S, Autenrieth J, Richter W
(See online at https://dx.doi.org/10.3205/16dkou403) - Cells for Cartilage Regeneration. In: Gimble JM, Marolt D, Oreffo R, Redl H, Wolbank S, editors. Cell Engineering and Regeneration. Cham: Springer International Publishing; 2018. p. 1-67
van Osch GJVM, Barbero A, Brittberg M, Correa D, Diederichs S, Goldring MB, et al.
(See online at https://doi.org/10.1007/978-3-319-37076-7_1-1) - Induzierte pluripotente Stammzellen als alternative Zellquelle für die Knorpelregeneration: Chondrogenese ohne unerwünschte Mineralisierungsaktivität Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2018)
Diederichs S, Hagmann S, Richter W
(See online at https://dx.doi.org/10.3205/18dkou544) - Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway. Front Cell Dev Biol. 2019;7(270): 270
Diederichs S, Klampfleuthner FAM, Moradi B, Richter W.
(See online at https://doi.org/10.3389/fcell.2019.00270) - Recent developments to utilize induced pluripotent stem cells for cartilage regeneration. 2019 TERMIS EU Abstract, eCM Periodical, 2019, Collection 3
Diederichs S
- The Role of Extracellular Matrix Expression, ERK1/2 Signaling and Cell Cohesiveness for Cartilage Yield from iPSCs. Int J Mol Sci. 2019;20(17)
Buchert J, Diederichs S, Kreuser U, Merle C, Richter W.
(See online at https://doi.org/10.3390/ijms20174295) - Ein initialer WNT-Puls verbessert Zellüberleben und Gewebeausbeute während der iPS-Chondrogenese. Deutscher Kongress fur Orthopädie und Unfallchirurgie (DKOU 2020), Zeitung für Orthopädie und Unfallchirurgie 5/20, 09.10.2020
Kreuser U, Richter W, Diederichs S
(See online at https://doi.org/10.1055/s-0040-1717236) - Initial WNT/β-Catenin Activation Enhanced Mesoderm Commitment, Extracellular Matrix Expression, Cell Aggregation and Cartilage Tissue Yield from Induced Pluripotent Stem Cells. Front Cell Dev Biol
Kreuser K, Buchert J, Haase A, Richter W, Diederichs S.
(See online at https://doi.org/10.3389/fcell.2020.581331) - Juveniler artikulärer Knorpel aus unbegrenzt verfügbaren induzierten pluripotenten Stammzellen. Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2020), Zeitung für Orthopädie und Unfallchirurgie 5/20, 09.10.2020
Diederichs S, Klamp euthner FAM, Richter W
(See online at https://doi.org/10.1055/s-0040-1717227) - Significance of MEF2C and RUNX3 Regulation for Endochondral Differentiation of Human Mesenchymal Progenitor Cells. Front Cell Dev Biol. 2020;8: 81. (IF 2019: 5.201)
Dreher SI, Fischer J, Walker T, Diederichs S, Richter W.
(See online at https://doi.org/10.3389/fcell.2020.00081)
