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A study released in STEM CELLS moves scientists a step closer to finding how to help the body regenerate joint cartilage ravaged by disease. Their work reveals a new method to quickly and efficiently produce virtually unlimited numbers of chondrocytes, the cells that form cartilage, from human skin cells converted to induced pluripotent stem cells (iPSCs). For the 54 million Americans suffering from arthritis - the nation's Number One disability - this could be great news.
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While a May 2018 report by Modern Healthcare says that currently over 1 million joint replacement surgeries occur every year in the United States alone -- and that number is expected to exceed 4 million by 2030 -- many medical researchers believe that the future of arthritis therapeutics lies in the application of stem cells to grow new joint cartilage (a process called "chondrogenesis"). Human iPSCs (hiPSCs) are a promising cell source for cartilage regenerative therapies and in vitro disease-modeling systems due to their pluripotency and unlimited proliferation capacity. Furthermore, iPSCs provide a means of developing patient-specific or genetically engineered cartilage to screen for osteoarthritis drugs.
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"That's why finding methods to rapidly and efficiently differentiate hiPSCs into chondrocytes in a reproducible and robust manner is critical," said Farshid Guilak, Ph.D., from Washington University's Center of Regenerative Medicine and Shriners Hospitals for Children (St. Louis, Mo.). He is a co-senior author of the study in STEM CELLS along with Charles A. Gersbach, Ph.D., from the Department of Biomedical Engineering at Duke University (Durham, N.C). Scientists from Cytex Therapeutics (Durham, N.C.) and Stanford University (Stanford, Calif.) also participated.
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Chondrocytes are the cells that produce and maintain the cartilage lining the surfaces of diarthrodial joints -- the free-moving types of joints you find, for example, in the hip and knee. "However," Dr. Guilak explained, "a disease like arthritis can destroy the cartilage in the joint and escalate inflammation. Ultimately, these changes lead to pain and loss of function that currently necessitates total joint replacement with an artificial prosthesis."
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In their study, the Guilak-Gersbach team demonstrated the development and application of a step-wise differentiation protocol validated in three unique and well-characterized hiPSC lines. They examined gene expression profiles and cartilaginous matrix production during the course of differentiation. To further purify committed chondroprogenitors, they used CRISPR-Cas9 genome engineering technology to knock-in a GFP reporter at the collagen type II alpha 1 chain (COL2A1) locus to test the hypothesis that purifying the chondroprogenitors could enhance articular cartilage-like matrix production.
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Most differentiation protocols to date have been based on trial-and-error delivery of growth factors without immediate consideration of the signaling pathways that direct and inhibit each stage of differentiation. Accordingly, chondrogenic differentiation is often dependent on the specific cell lines used, and broad application of iPSC chondrogenesis protocols has not been independently demonstrated with multiple cell lines and in multiple laboratories.
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Recently, critical insights from developmental biology have elucidated the sequence of signaling pathways needed for PSC lineage specification to a number of cell fates.
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"By reproducing these reported signaling pathways in vitro, in combination with existing chondrogenic differentiation approaches, we sought to establish a rapid and highly reproducible protocol for hiPSC chondrogenesis that is broadly applicable across various hiPSC lines," said Chia-Lung Wu, Ph.D., the co-first author of the study. "Since hiPSC differentiation processes are inherently unpredictable and can often produce heterogeneous cell populations over the course of differentiation, an important goal of differentiation protocols is to minimize variability in hiPSC differentiation potential, which may arise from characteristics of the donor and/or reprogramming method. Therefore, we hypothesized that purifying the committed chondroprogenitors would improve hiPSC chondrogenesis."
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The method obtained the desired results.
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"The purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations," Dr. Shaunak Adkar, Ph.D., co-first author of the study reported. "The development of processes for rapid and repeatable induction of iPSCs into joint cell tissue will hopefully enable the identification of novel therapies for joint diseases such as osteoarthritis."
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Jan Nolta, Ph.D., Editor-in-Chief of STEM CELLS, said, "The elegant techniques used by the the Guilak-Gersbach team generated improved numbers of pure chondroprogenitors, a step that was crucially needed to propel the promising field of stem cell- mediated cartilage repair forward."
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This work was supported in part by the Arthritis Foundation, the Nancy Taylor Foundation for Chronic Diseases, and the National Institutes of Health.
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The full article, "Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing" can be accessed at https://stemcellsjournals.onlinelibrary.wiley.com/doi/abs/10.1002/stem.2931.
