Cartilage tissue engineering identifies abnormal human induced pluripotent stem cells

Yamashita, Akihiro; Liu, Shiying; Woltjen, Knut; Thomas, Bradley; Meng, Guoliang; Hotta, Akitsu; Takahashi, Kazutoshi; Ellis, James; Yamanaka, Shinya; Rancourt, Derrick E.

doi:10.1038/srep01978

Cited by 44 publications

(30 citation statements)

References 24 publications

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“…Indeed, the heterogeneity of in vitro chondrogenesis may preclude our understanding of the mechanisms by which genetic variation can impact cartilage development and arthritis progression. Previous attempts to purify iPSC-derived CPs relied on many rounds of sorting multiple surface markers, using reporter constructs that are activated by minimal promoter regions of chondrocyte markers, or physical dissection of self-aggregating chondroprogenitors [20,29,37,49,50]. During chondrocyte differentiation, COL2A1 expression is regulated by concerted coordination of promoter regions, distal and intronic enhancers, and UTRs [51–53].…”

Section: Discussionmentioning

confidence: 99%

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

et al. 2018

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The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations. The ability to enrich for chrondroprogenitors and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications.

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Section: Discussionmentioning

confidence: 99%

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

et al. 2018

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“…60 Efforts to derive iPSCs from adult chondrocytes using nonintegrating methods such as mRNA delivery may address some of these concerns, 61 and methods to isolate successfully differentiated cells can add additional control over the phenotype of cells transplanted for therapy. 62,63 Indeed, the multi-step differentiation strategy employed in this study uses positive selection with a Col2 reporter as an additional safeguard to exclude nondifferentiated iPSCs.…”

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confidence: 99%

Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

Diekman

Thakore²,

O’Connor³

et al. 2015

Tissue Engineering Part A

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The limited regenerative capacity of articular cartilage contributes to progressive joint dysfunction associated with cartilage injury or osteoarthritis. Cartilage tissue engineering seeks to provide a biological substitute for repairing damaged or diseased cartilage, but requires a cell source with the capacity for extensive expansion without loss of chondrogenic potential. In this study, we hypothesized that decreased expression of the cell cycle inhibitor p21 would enhance the proliferative and chondrogenic potential of differentiated induced pluripotent stem cells (iPSCs). Murine iPSCs were directed to differentiate toward the chondrogenic lineage with an established protocol and then engineered to express a short hairpin RNA (shRNA) to reduce the expression of p21. Cells expressing the p21 shRNA demonstrated higher proliferative potential during monolayer expansion and increased synthesis of glycosaminoglycans (GAGs) in pellet cultures. Furthermore, these cells could be expanded *150-fold over three additional passages without a reduction in the subsequent production of GAGs, while control cells showed reduced potential for GAG synthesis with three additional passages. In pellets from extensively passaged cells, knockdown of p21 attenuated the sharp decrease in cell number that occurred in control cells, and immunohistochemical analysis showed that p21 knockdown limited the production of type I and type X collagen while maintaining synthesis of cartilage-specific type II collagen. These findings suggest that manipulating the cell cycle can augment the monolayer expansion and preserve the chondrogenic capacity of differentiated iPSCs, providing a strategy for enhancing iPSC-based cartilage tissue engineering. IntroductionA rticular cartilage provides a low-friction loadbearing surface in diarthrodial joints such as the knee and hip.1 However, cartilage degeneration or loss that occurs with osteoarthritis (OA) is associated with significant pain and joint dysfunction. 2 The risk for cartilage degeneration is enhanced by the presence of focal damage, 3,4 prompting efforts to treat cartilage defects using techniques such as marrow stimulation.5 Using a combination of cells, scaffolds, and growth factors to engineer cartilage for transplantation has been proposed as a potential therapy, but the optimal cell source has yet to be identified. 6 The use of autologous chondrocytes requires an additional procedure to harvest healthy cartilage and follow-up studies have indicated the presence of suboptimal fibrocartilage tissue after repair.7 Adult stem cells also have limitations, as bone marrow-derived mesenchymal stem/stromal cells (MSCs) display a propensity for mineralization 8,9 and adiposederived stem cells (ASCs) may need additional growth factors for full chondrogenesis in some systems.10,11 Embryonic stem cells and induced pluripotent stem cells (iPSCs) have emerged as other alternatives, but require extensive differentiation protocols to avoid a remnant of undifferentiated cells with tumor-forming potential....

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“…Another report on 21 human iPSC lines and 6 human ESC lines indicated that certain human iPSC clones were in a pro-oncogenic state, as shown by the ectopic presence of secretory tumor tissue during in vitro cartilage differentiation [109] . These reports together implied that a marker foreseeing pro-oncogenic differences in iPSC lines would be required for establishing safe therapy using iPSCs.…”

Section: Selecting the Most Appropriate Ipsc Line For Clinical Usementioning

confidence: 99%

Methods of induced pluripotent stem cells for clinical application

Seki

Fukuda

2015

WJSC

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Reprograming somatic cells using exogenetic gene expression represents a groundbreaking step in regenerative medicine. Induced pluripotent stem cells (iPSCs) are expected to yield novel therapies with the potential to solve many issues involving incurable diseases. In particular, applying iPSCs clinically holds the promise of addressing the problems of immune rejection and ethics that have hampered the clinical applications of embryonic stem cells. However, as iPSC research has progressed, new problems have emerged that need to be solved before the routine clinical application of iPSCs can become established. In this review, we discuss the current technologies and future problems of human iPSC generation methods for clinical use. Core tip: Each induced pluripotent stem cells methodology has advantages and disadvantages, as in the case of autologous vs allogenic transplantation, and the choice of appropriate strategy may vary depending on the intended use. Additionally, to avoid tumorigenesis and to establish effective differentiation into the intended cells, further investigation is needed to identify the most suitable iPSC line and how these lines should be selected.Seki T, Fukuda K. Methods of induced pluripotent stem cells for clinical application.

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Cartilage tissue engineering identifies abnormal human induced pluripotent stem cells

Cited by 44 publications

References 24 publications

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing

Knockdown of the Cell Cycle Inhibitor p21 Enhances Cartilage Formation by Induced Pluripotent Stem Cells

Methods of induced pluripotent stem cells for clinical application

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