Osteoarthritis is a progressive disease where the protective layer of articular cartilage that cushions the junctional surface of bones in joints degenerates. This restricts movement and causes chronic pain. Joint replacement surgery offers the only permanent solution once articular cartilages erode.
Researchers are developing a stem cell–based, cryopreservable bioimplant that can repair articular cartilage before arthritis sets in. They have demonstrated its efficacy in animal models. Translation of this treatment into the clinics is currently underway with a $6 million grant from the California Institute of Regenerative Medicine.
Denis Evseenko, MD, PhD, associate professor of orthopedic surgery and vice chair for research of orthopedic surgery at the Keck School of Medicine in the University of Southern California (USC), has been spearheading this quest for over a decade. In an exclusive interview with GEN, Evseenko discusses the challenges in developing this promising treatment for osteoarthritis.
Delving into differentiation
Evseenko’s team has shown that defective articular cartilage in synovial joints can be restored to full thickness, long term, in pigs using cartilage cells (chondrocytes) derived from human embryonic stem cells (hESCs). The bioimplant builds on an in-depth understanding of cartilage development that his team has published in PNAS, Stem Cell Reports, Nature Communications, and Nature Regenerative Medicine.
A challenge in culturing articular chondrocytes is the progressive restriction of developmental ESC fate. “It sounds simple,” he says, “but it’s not easy to recapitulate normal embryogenesis and instruct cells to become a particular cell type.”
Understanding cell fate decisions is not enough. One needs to ensure that the desired cell fate predominates. Conventional protocols for differentiating ESCs result in multiple cell types. “We spent the first three to five years trying to understand how to differentiate ESCs into articulate cartilage,” Evseenko continues.
Articular cartilage is soft enough to enable movement but strong enough to prevent damage to the underlying bone. Most tissues consist primarily of cells, but articular cartilage is 95% extracellular matrix (ECM), a hydrogel of proteins such as collagen.
“We succeeded in making highly committed cells, but it took us some time to convince the scientific community that these cells are the same as endogenous articular chondrocytes,” Evseenko notes. His team compared endogenous chondrocytes to chondrocytes derived in a dish from hESCs.
“I was surprised. The molecular and cellular identity of hESC-derived chondrocytes is similar to that of human fetal chondrocytes—potent, anabolic, proliferative, and capable of producing a lot of ECM,” he tells GEN. (Adult chondrocytes, once damaged, do not rebuild.) He adds, “Physical parameters, gene expression, signaling networks, and proteins produced by endogenous primary cells look similar to chondrocytes made from stem cells in the dish.”
Advantages of a scaffold
After deriving articular chondrocytes from hESCs in vitro, Evseenko’s team investigated the benefits of these cells. The team generated focal lesions in articular cartilages in live pigs and tested the cells at multiple doses and with different carriers. Assessments included the direct injection of free articulate chondrocytes and the embedding of articulate chondrocytes in a collagen membrane scaffold. Membrane-embedded transplantation gave the best results and was developed into the Plurocart bioimplant. The pig-collagen membrane is a clinically approved scaffold manufactured by Vericell that is currently used in dentistry.
“The therapy stops arthritis before it begins by targeting a defect in an overall healthy joint,” Evseenko says. Once degeneration sets in, the only option is joint replacement. In addition to structural integrity and cellular retention, an advantage of transplanting cells on a scaffold is the co-transfer of abundant ECM.
“We seed membranes with stem cell–derived chondrocytes and let it mature in the dish for up to two weeks to allow the cells to produce this biogenically active matrix,” Evseenko continues. The ECM is a depot of growth factors, mitogens, and other signaling and structural molecules.
The team found implanted human chondrocytes integrated into articular cartilage lesions in pigs at the end of the six months. “These exogenous implanted cells secrete factors that induce an endogenous regenerative response in host cells,” Evseenko explains. “The pig cells within the defects also started to differentiate into cartilage upon implantation. We didn’t see that in controls. It’s not the injury but the implant that does it.” The restored articular cartilage was composed of human and induced pig cells that became articular cartilage. This indicates a combined effect of direct differentiation and paracrine induction. The Evseenko team identified a few factors that play a role in the process.
A lasting and scalable product
Once developed, the bioimplant will be an allogeneic, off-the shelf product for clinical use. Evseenko points out, “You can manufacture large quantities of these implants. We also developed a cryopreservation condition in which this implant can be stored indefinitely.” Cryopreservation of the implant enables transplant surgeries at remote locations, precluding the need for distributed, time-bound manufacturing to achieve proximity to the site of implementation and the date of the surgery. This reduces overall costs and increases the product’s practical relevance.
In addition, scaling the production of the implants is easy,” Evseenko says. “We can produce hundreds of implants easily already without any additional facilities or reagents. It’s just a matter of modular expansion.”
Scaling to attain commercialization goals will require process development for batch production, getting the product to GMP compliance, meeting release criteria, and optimizing preservation. The team expects to achieve cost reduction in the processing and to have a product that will be extremely cheap. “At this point, we’re past all critical steps required for scaling up,” Evseenko says. “It’s already at an industrial grade.”
Allogeneic products derived from individuals other than the patient offer an obvious advantage over autologous products that are generated from the patient’s own cells. However, allogeneic products run the risk of triggering an immune response. Cartilage does not include blood vessels or immune cells, and therefore does not reject allogeneic material, providing a distinct advantage.
“It is a common practice to take fragments of articular cartilage or even osteo-chondro allografts from one person and place it in another person,” Evseenko continues. “There’s no immunological rejection, like you would have in a liver or kidney transplant. This is a huge advantage and a big deal for a clinical therapy. We don’t need immunosuppression.”
This immune advantage could rationally favor xenotransplantation approaches where chondrocytes derived from pig embryonic stem cells are implanted into human joints without immune rejection. There’s only one problem to that. So far, nobody has been able to create pig pluripotent embryonic stem cells.
For therapeutic purposes, Evseenko prefers an allogeneic over a xenogeneic product: “When you transplant from one human to another, even if it’s biologically mismatched, the degree of this mismatch is relatively minor. When you transplant cells from one species to another, you multiply this immunological mismatch many times. So even if relevant pig cells existed, we would not go for it.”
One of the bottlenecks in the production of the Plurocart implant could be acquiring hESCs as source material. A potential alternative could be the ability to differentiate induced pluripotent stem cells (iPSCs) into chondrocytes. However, Evseenko does not consider using iPSCs a viable option.
“Ethical issues forced people to work primarily with iPSCs; however, iPSCs are usually produced by artificial manipulation of the genome and integration of factors that can be detrimental and break down the regulatory machinery in cells. It’s an artificial, human-made cell type, which has not really been studied,” he says. “I’m conservative. I care less than maybe others about the ethical aspect of hESCs. From the biological perspective, they’re more natural and potent.”
Challenges in regulated differentiation
“Our skeleton starts out as cartilage and ossifies during development,” Evseenko explains. Only around 3% remains permanently as cartilage in articular joints. Therefore, a challenge in generating articular cartilage from ESCs is to stop differentiation at a critical juncture where the chondrocytes are neither stem cells nor bone.
“We spent a lot of time trying to understand what pushes chondrocytes into this articular cartilage cell fate and found that the key factor expressed in developing joints is leukemia inhibitory factor (LIF).”
Previous studies have shown LIF prevents terminal differentiation of different cell types and is required to maintain pluripotent stem cells, progenitors, and immature cells in culture. Developing joints are flooded with LIF to preserve cartilage cells from becoming bone.
“We applied LIF in our protocols. We’ve demonstrated if we delete the LIF receptor in mice, or if we delete some of the critical pathways downstream of LIF, we lose cartilage stem cells,” Evseenko tells GEN.
The team has shown that the gp130/Stat3 pathway downstream of LIF is critical for cartilage development. Deleting Stat3 in postnatal chondrocytes results in progressive dysfunction of the articular cartilage and their differentiation into bone, indicating Stat3 controls the switch. The application of basic developmental insights enabled Evseenko’s team to preserve articular chondrocytes in the bioimplant.
Producing a cell therapy product requires stringent quality control tests to demonstrate the potency and purity of the product. These must ensure that external reagents and contaminants have been completely removed and that stem cells used as source materials are entirely differentiated.
“We have presented the technology to the FDA and had a particularly good and positive feedback. Our technology requires magnetic sorting of chondrocyte progenitors as a purification step early in the development process,” Evseenko says. ”It eliminates all contaminating cell populations, including residual pluripotent cells. We’ve also conducted single-cell sequencing as part of the assessment. The purity of this product was phenomenal. We had more than 97% articular chondrocytes in the final product.”
Evseenko finds magnetic sorting more clinically applicable than flow cytometry-based purification approaches. “We use an automated Miltenyi platform called CliniMACs. The company has successfully developed GMP compatible, magnetic sorting devices and reagents for human clinical trials that are used in CAR T-cell therapy.”
The team will use the grant from the California Institute of Regenerative Medicine to develop GMP-compliant standard operating procedures. The team will then manufacture 64 implants that will be GMP-released and cryopreserved for first-in-human trials. The implants will be used to treat large and small defects. Evseenko plans to submit an investigational new drug application and the updated clinical trial protocol to the FDA within the next couple of years.