Tissue engineered cartilage from autologous, dermis-isolated, adult, stem (DIAS) cells

Cartilage degeneration resulting from injuries or wear-and-tear leads to osteoarthritis, which impacts millions and costs in excess of $65B per annum. No long-term solutions exist for cartilage degeneration, but cellular therapies hold promise toward replacing degenerated cartilage with healthy tissue. This Development Candidate Feasibility Award is a first step toward the overall goal of developing a cell-based cartilage repair therapy using stem cells derived from the skin. The therapy would consist of using a skin biopsy to harvest dermis-isolated, adult stem cells (DIAS cells), which will undergo processing to yield neocartilage. This neocartilage will then be implanted into the patient’s joint to restore or improve mobility. Work during this progress report period has been divided into project preparation and scientific progress. Project preparation includes setting up facilities and approvals for work with human DIAS cells, identifying sources and acquiring human skin for DIAS cell isolation, and hiring and training personnel. Scientific progress includes a publication on co-cultures using stem cells, work on culturing larger numbers of cells using low oxygen tension, comparing stem cells from human skin of different anatomical locations, and gaining an understanding of the niches where skin stem cells may reside. The project now has a consistent source of human dermis tissue from which stem cells can be isolated. This includes skin containing hair follicles and also skin without follicles. Spherical culture of human skin-derived stem cells has been performed. It was found that directing stem cells into cells that make cartilaginous matrix can be more efficacious if done under low oxygen tension. Since much of the prior work on directing stem cells from the skin to form neocartilage has been done using animal-derived stem cells, in the next project period neocartilage will be formed using human stem cells instead. Technologies developed using animal models can thus be translated toward human use.

Resulting from injuries or wear-and-tear that leads to osteoarthritis, cartilage degeneration is a problem that costs in excess of $65B per annum. Toward developing a long-term solution for this vexing problem, cellular therapies hold the promise of replacing degenerated cartilage with healthy tissue. This Development Candidate Feasibility Award is a first step toward the overall goal of developing a cell-based cartilage repair therapy using stem cells derived from the skin. The therapy would begin with a biopsy of the patient’s own skin to harvest dermis isolated, adult stem cells (DIAS cells), which will undergo processing to yield neocartilage. This neocartilage will then be implanted into the patient’s joint to restore or improve mobility. During this progress report period, a major milestone has been completed. Previously, DIAS cells have been isolated from various animal models, including sheep, goat, and rabbit. Comparing animal skin and human skin showed notable differences, including morphology, response to enzymatic digestion, and the rate at which cells attach to tissue culture plastic. As a result, protocols that successfully yielded DIAS cells using animal models could not be directly applied to isolating DIAS cells from human skin. During the first six months of this reporting period, human DIAS cells were isolated and used to engineer neocartilage. Characterizing the human DIAS cell population showed that cells shared similar characteristics with stem and progenitor cells previously identified by other groups as originating from various niches of the skin. Neocartilage constructs formed using human DIAS cells were found to contain twice as much glycosaminoglycans and three times more collagen; these are cartilage extracellular matrix component important in imparting mechanical function to the tissue. Neocartilage constructs generated from human DIAS cells also contained five times higher compressive modulus and close to twice the tensile modulus of constructs generated using sheep DIAS cells. The completion of this important milestone allowed for progression to the next milestones of this award. Milestone 2 of this award consists of examining safety of the engineered constructs in a small animal model. For the scaling-up of constructs to be used in an athymic mouse study, an experiment was conducted to finalize our protocol for generating human DIAS cell constructs, using what have been learned both from Milestone 1 and also from literature sources. Three methods for generating neocartilage constructs were examined. While the resultant constructs did not differ in mechanical properties, one method nonetheless yielded constructs of more uniform size and greater cell and glycosaminoglycan content. For milestones 3 and 4, the originally proposed studies were to implant autologous neocartilage constructs in intermediate and large animal studies to examine efficacy of repair. To improve the translational potential of this project, CIRM has requested that neocartilage constructs of human origin be used instead. To ensure that the implanted constructs are not rejected, progress during this reporting period also includes identifying methods to immunosuppress intermediate and large animal models. To summarize, progress during this reporting period includes the completion of Milestone 1 and work toward all other milestones of this award. Additionally, two papers have been published thus far to disseminate scientific findings to the public.

Articular cartilage injuries and degeneration eventually lead to osteoarthritis, costing $65B per year and affecting over 25% of adults over the age of 65. This Development Candidate Feasibility (DCF) Award seeks to address cartilage defects classified by the International Cartilage Repair Society (ICRS) as grades II-IV with the intent of halting the progression of cartilage damage before it creates arthritic changes in joint compartments. The therapy would begin with a biopsy of the patient’s own skin to harvest dermis isolated, adult stem cells (DIAS cells), which will undergo processing to yield neocartilage. This neocartilage will then be implanted into the patient’s joint to restore or improve mobility. The project objective has not changed from previous progress reports, though new milestones have been proposed during this reporting period and approved by CIRM, as described below. Milestone 1 was completed and, after reporting of preliminary data from Milestone 2, a prior approval request was submitted with a description of critical path experiments to amend the milestones, and a no cost extension was also submitted. These have been approved by CIRM. The new, remaining milestones to be completed consist of: three studies in the athymic mice using human DIAS neocartilage generated from foreskin, breast skin, and abdominal skin, from multiple donors (Milestone 2); a study to apply aggregate redifferentiation to human DIAS cells to yield cells with a chondrogenic phenotype (Milestone 3), and the rabbit study as initially approved, using human DIAS cells and with an abridged timeline (Milestone 4). As proposed and approved, the three athymic mouse studies are currently in progress. The mouse portion of the first study, using breast skin-derived DIAS cells, has been completed. Studies using abdominal skin- and foreskin-derived DIAS cells are on track for completion in March and April, 2016. The aggregation redifferentiation study is currently ongoing, and neocartilage constructs have yet to be generated. The rabbit study is under planning for its immunosuppression component. Additionally, a paper was published during this reporting period to disseminate scientific findings to the public.

This Development Candidate Feasibility (DCF) Award seeks to address cartilage defects classified by the International Cartilage Repair Society (ICRS) as grades II-IV with the intent of halting the progression of cartilage damage before it creates arthritic changes in joint compartments. The medical significance of this project is that articular cartilage injuries and degeneration eventually lead to osteoarthritis, which costs $65B per year and affects over 25% of adults over the age of 65. The envisioned therapy would begin with a biopsy of the patient’s own skin to harvest dermis isolated, adult stem cells (DIAS cells), which would undergo processing to yield neocartilage. This neocartilage would then be implanted into the patient’s joint to restore or improve mobility. Milestone 1 was completed, and, after reporting of preliminary data from Milestone 2, a prior approval request was submitted and approved with revised milestones. These, too, have now been completed. For Milestone 2, three studies in the athymic mice using human DIAS neocartilage generated from foreskin, breast skin, and abdominal skin, from multiple donors were conducted to determine whether variability existed due to anatomical location and different donors. The goal was to determine whether engineered constructs would maintain stability, integrity, and viability in vivo without eliciting adverse effects. Anatomical and donor variability were found, with foreskin-derived human DIAS cells displaying constructs with the lowest level of donor variability; all of the foreskin-derived constructs retained stability, integrity, and viability. Regardless of anatomical location and donors, no DIAS cell-derived constructs elicited adverse host responses. Milestone 3 consisted of a study to apply aggregate redifferentiation to human DIAS cells to yield cells with a chondrogenic phenotype. The aggregate redifferentiation technique, previously optimized in animal models, has also shown efficacy with human marrow-derived mesenchymal stem cells. Applied to constructs formed using human foreskin-derived DIAS cells, aggregate redifferentiation was shown to be efficacious in improving the biochemical and biomechanical properties of the constructs. Specifically, the constructs contained more cells and extracellular matrix. The compressive properties of constructs that had been formed with aggregate redifferentiated human DIAS cells were also higher than those of controls. Milestone 4 consisted of determining whether over 75% of the engineered constructs would retain mechanical integrity after orthotopic implantation in a rabbit model. The mechanical properties of the engineered constructs were not sufficiently high to survive cycling within rabbit knees ex vivo after being placed in cartilage defects. Additional work needs to be performed to strengthen the human DIAS cell constructs for implantation; the goal would be to attain the mechanical properties currently achievable with differentiated cartilage cells derived from animal models.