CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line

Yang, Maozhou; Zhang, Liang; Stevens, Jeff; Gibson, G. Edward

doi:10.1016/j.bone.2014.09.005

Cited by 27 publications

(19 citation statements)

References 27 publications

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“…The RCS-Cas9 cells, that co-express Cas9 and mCherry genes [13], were a generous gift of Dr. Gary Gibson (Henry Ford Hospital). Both RCS line chondrocytes were cultured as high density monolayers (2.0 × 10 6 cells/ cm 2 ) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; Hyclone) and 1% L-glutamine and penicillin-streptomycin, and incubated at 37° C in a 5% CO 2 environment.…”

Section: Methodsmentioning

confidence: 99%

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CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

et al. 2016

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Hyaluronan (HA) plays an essential role in cartilage where it functions to retain aggrecan. Previous studies have suggested that aggrecan is anchored indirectly to the plasma membrane of chondrocytes via its binding to cell-associated HA. However, reagents used to test these observations such as hyaluronidase and HA oligosaccharides are short term and may have side activities that complicate interpretation. Using the CRISPR/Cas9 gene editing approach, a model system was developed by generating HA-deficient chondrocyte cell lines. HA synthase-2 (Has2)-specific single guide RNA was introduced into two different variant lines of rat chondrosarcoma chondrocytes; knockout clones were isolated and characterized. Two other members of the HA synthase gene family were expressed at very low relative copy number but showed no compensatory response in the Has2 knockouts. Wild type chondrocytes of both variants exhibited large pericellular matrices or coats extending from the plasma membrane. Addition of purified aggrecan monomer expanded the size of these coats as the proteoglycan became retained within the pericellular matrix. Has2 knockout chondrocytes lost all capacity to assemble a particle-excluding pericellular matrix and more importantly, no matrices formed around the knockout cells following the addition of purified aggrecan. When grown as pellet cultures so as to generate a bioengineered neocartilage tissue, the Has2 knockout chondrocytes assumed a tightly-compacted morphology as compared to the wild type cells. When knockout chondrocytes were transduced with Adeno-ZsGreen1-mycHas2, the cell-associated pericellular matrices were restored including the capacity to bind and incorporate additional exogenous aggrecan into the matrix. These results suggest that HA is essential for aggrecan retention and maintaining cell separation during tissue formation.

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Section: Methodsmentioning

confidence: 99%

“…In 2014, Yang et al . [13] developed a Cas9 stable transfectant clone of a different RCS cell line and used these cells to generate a CRISPR/Cas9 mediated knockout of aggrecan. We obtained the wild type parental RCS cells (labeled in this study as RCS-Cas9).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

et al. 2016

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“…Osteogenic determination was performed with alizarin red S with Mayer's hematoxylin staining and with Von Kossa with nuclear fast red staining method to show calcium deposits seen in osteocytes (12,31,32). Chodrogenic determination was done with safranin O staining, which stains cartilaginous tissue, and immunostained with an aggrecan antibody, a proteoglycan highly expressed in cartilage tissue (12,33,34). The immunostained chodrogenic cells were imaged at 200x on a DSU Spinning Disk Confocal Microscope (Olympus, Center Valley, PA).…”

Section: Msc + Minocycline-loaded Hydrogel + Sa Co-culture and Characmentioning

confidence: 99%

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes

Guerra

Cantu

Vecchi

et al. 2015

AAPS J

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Abstract. Mesenchymal stromal/stem cells (MSCs) have demonstrated favorable wound healing properties in addition to their differentiation capacity. MSCs encapsulated in biomaterials such as gelatin and polyethylene glycol (PEG) composite hydrogels have displayed an immunophenotype change that leads to the release of cytokines and growth factors to accelerate wound healing. However, therapeutic potential of implanted MSC-loaded hydrogels may be limited by non-specific protein adsorption that facilitates adhesion of bacterial pathogens such as planktonic Staphylococcus aureus (SA) to the surface with subsequent biofilm formation resistant to immune cell recognition and antibiotic activity. In this study, we demonstrate that blood-derived primary leukocytes and bone marrow-derived MSCs cannot inhibit colony-forming abilities of planktonic or biofilm-associated SA. However, we show that hydrogels loaded with MSCs and minocycline significantly inhibit colony-forming abilities of planktonic SA while maintaining MSC viability and multipotency. Our results suggest that minocycline and MSC-loaded hydrogels may decrease the bioburden of SA at implant sites in wounds, and may improve the wound healing capabilities of MSC-loaded hydrogels.

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“…In one example, gRNAs were designed to knockout hyaluronan synthase 2 ( Has2 ) in a rat chondrosarcoma (RCS) line constitutively expressing Cas9 [118] [119]. Has2 is an enzyme that generates hyaluronan , and whose loss of function is an embryonic lethal in mice.…”

Section: Disease Modeling Applicationsmentioning

confidence: 99%

“…Has2 is an enzyme that generates hyaluronan , and whose loss of function is an embryonic lethal in mice. [118] [119] By editing Has2 in the chondrosarcoma line, Has2 was shown to retain aggrecan (an important proteoglycan of articular cartilage) in the pericellular matrix of mature chondrocytes [118] [119]. Thus, genome engineering can be applied to tissue engineering platforms to both replicate knockout studies in animal models and further elucidate protein function when traditional in vivo knockout is cumbersome.…”

Section: Disease Modeling Applicationsmentioning

confidence: 99%

Genome Engineering for Personalized Arthritis Therapeutics

Adkar

Brunger

Willard

et al. 2017

Trends in Molecular Medicine

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Arthritis represents a family of complex joint pathologies responsible for the majority of musculoskeletal conditions. Nearly all diseases within this family, including osteoarthritis, rheumatoid arthritis, and juvenile idiopathic arthritis, are chronic conditions with few or no disease-modifying therapeutics available. Advances in genome engineering technology, most recently with CRISPR-Cas9, have revolutionized our ability to interrogate and validate genetic and epigenetic elements associated with chronic diseases such as arthritis. These technologies, together with cell reprogramming methods, including the use of induced pluripotent stem cells, provide a platform for human disease modeling. We summarize new evidence from genome-wide association studies and genomics that substantiates a genetic basis for arthritis pathogenesis. We also review the potential contributions of genome engineering in the development of new arthritis therapeutics.

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CRISPR/Cas9 mediated generation of stable chondrocyte cell lines with targeted gene knockouts; analysis of an aggrecan knockout cell line

Cited by 27 publications

References 27 publications

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

Mesenchymal Stromal/Stem Cell and Minocycline-Loaded Hydrogels Inhibit the Growth of Staphylococcus aureus that Evades Immunomodulation of Blood-Derived Leukocytes

Genome Engineering for Personalized Arthritis Therapeutics

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