Special Report on StemCells/Cell Therapy: Skin in the game

Cell therapy meets gene therapy to advance dermatology

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Skin in the game
Cell therapy meets gene therapy to advance dermatology
By Randall C Willis
Thursday, July 13; an unremarkable evening in the city of London.
Late in the evening, people walk the streets, largely going about their lives as anyone does on a Thursday night, perhaps thinking about weekend plans with family and friends.
Vehicles of all shapes pass down the roadways of East London: cars, trucks, motorbikes, mopeds. No one even notices.
10:25 pm: The consistent hum of the evening is shattered with a scream as a man clutches his face in agony. Mopeds roar away.
24 minutes later, another scream. Another 16 minutes, another scream. Another, 13 minutes later. And yet another, 19 minutes after that.
Five lives irreparably changed over a 72-minute period. Each one a victim of an acid attack.
Terrorism? Robbery? Personal vendetta?
None of that is important for the five people whose clothes are burning, whose skin is melting under the chemical assault.
Right now, they need medical attention, and for some if not all, they need skin grafts to repair the damage done this terrible night.
Patching the damage
Whether the result of accidental burning, such as with chemical exposure, fire or explosions, or the result of clinical pathologies, such as diabetic ulceration, skin wounds continue to be a large focus of cell-based therapies, with recent developments attending to the injuries in situ.
Although conventional skin grafting, involving removing skin from one part of the body to cover another part, is still common, the last decade or so has seen a large-scale push into in-vitro-sourced skin replacement, including materials generated from human placenta (e.g., Grafix), amnion (e.g., AmnioExcel, EpiFix) or tissues taken from cadavers (e.g., ApliGraf, TheraSkin). (See also the article “Regenerating interest in stem cell medicine” in the August 2012 issue of DDNews.)
In a 2016 review, Alex Kong and colleagues as University of Southern California’s Keck School of Medicine suggested more than four million people in the United States are impacted by chronic wounds at an annual cost of $50 billion for treatment. When you include other sources of skin ulceration and wounding, the number of patients may top 150 million.
“Overall, the efficacy of cell-based wound dressings appears to cover a broad range of indications, and since most of these dressings have been available for 10 years or less, continued research is necessary to evaluate whether cell-based dressings could potentially replace the current [standard-of-care] altogether,” the authors concluded.
Using this success as a jumping off point, however, many other groups are starting to move away from such tissue-based efforts and toward methods that apply cells directly to the wound area, stimulating the natural healing functions that have been otherwise dormant in the wound fringes.
RenovaCare, for example, has developed the CellMist system whereby stem cells are isolated from a small sample of a patient’s skin and then gently sprayed onto the injured site in a saline suspension. Because the stem cells come from the patient him or herself, there is no risk of rejection as might be the case with other grafting options.
In April, the company reported on the efficacy of CellMist in the treatment of a variety of burn injuries.
“In the case of one patient with severe electrical burns to over one-third of his body, his wounds were sprayed with 23 million stem cells isolated from a tiny 2”-by-2” sample of his own skin,” recounted President and CEO Thomas Bold in the announcement. “Within five days of treatment, his chest and arms were already healed. Four days later, the patient was discharged from the hospital.”
Similarly, Tae Hyun Choi and colleagues at Seoul National University recently described their efforts to convert human adipose-derived stem cells (hADSCs) into fibroblasts to facilitate wound healing in a mouse model.
Using FACS, western blotting, RT-PCR and immunohistochemistry, the researchers showed that when hADSCs were cultured in human fibroblast-conditioned medium (F-CM), the stem cells demonstrated several fibroblast-specific markers, including dramatically increased levels of type 1 pro-collagen. As well, when transplanted to injured nude mice, the treated cells integrated into the surrounding skin and promoted accelerated wound contraction and re-epithelialization.
“Our group aimed to differentiate F-CM-treated hADSCs into fibroblast-like cells and confirmed the efficiency of differentiated cells in promoting collagen type I synthesis in a wound healing model,” the authors concluded. “With potential applications in the clinic, our study is the first research of its kind with differentiated hADSCs being applied for treating full-thickness skin wounds.”
Unfortunately, whereas accidents involving burns or related wounds can be somewhat avoided, skin wounds are a normal hazard of life for a select group of individuals. For people with epidermolysis bullosa (EB), for example, the simple act of putting on a shirt can see their skin detach from their bodies.
Skin tight
“Although EB is a rare disease, with an estimated incidence of one in 20,000 people, this equates to a worldwide incidence of approximately 500,000 individuals,” wrote John McGrath and colleagues from King’s College London in 2014. “Collectively, this population has a desperate need for innovative therapies that reduce disease burden, improve quality of life and make advances toward a cure.”
EB is a family of skin disorders that typically involve problems in the dermal-epidermal junction where deficiencies in structural elements like type 1 collagen, which effectively act as a cellular adhesive, cause blisters to form even with the most minor trauma.
“Recently, considerable progress has been made in developing new treatments for EB, including gene, protein and drug therapy, although cell therapies have perhaps shown the most clinical translation,” the authors suggested. “The major challenge is to restore an intact epithelium and provide some protection against mechanical trauma. To that end, therapeutic use of autologous or allogeneic cells, used locally or systemically, is now being explored in clinical trials.”
One such example is from Holostem Terapie Avanzate, a company that is using corrective gene therapy in epidermal stem cells to treat patients with junctional EB.
Earlier this year, Holostem Scientific Director Michele De Luca and R&D Director Graziella Pellegrini and colleagues from several universities described their efforts to use laminin-322-corrected keratinocytes from a patient with junctional EB to heal a long-term lesion.
Cells were grown into cohesive epidermal sheets and transplanted to the leg ulcer, completely engrafting within two weeks. At follow-up, the skin looked perfectly normal and demonstrated a normal dermal-epidermal junction.
“Because human epidermis is renewed monthly, the patient’s transgenic epidermis underwent at least 16 complete renewal cycles during the 16 months of follow-up,” the authors offered. “Thus, the long-term maintenance of the regenerated epidermis must be due to the engraftment of self-renewing transduced epidermal stem cells.”
They used quantitative real-time RT-PCR and Southern blot analysis to confirm that the entire regenerated epidermis was the product of engrafted stem cells.
Many of the same researchers have also recently shown they can repeat this process in preclinical studies of recessive dystrophic EB (RDEB) with gene therapy involving the gene for type VII collagen (COL7A1), and the company has recently initiated a Phase 1/2 clinical trial.
In collaboration with Intrexon, Fibrocell is taking a similar approach as Holostem, but in this case the genetic modification involves COL7 in autologous fibroblasts, which are then injected the cells into the wounds of patients with RDEB.
In June, Fibrocell announced it had completed dosing of the first cohort of patients in its Phase 1/2 trial.
Korea’s Anterogen, meanwhile, has initiated a Phase 1/2 clinical study of hydrogel-based sheets of allogeneic adipose-derived mesenchymal stem cells in the treatment of RDEB. The cells, the company suggests, have anti-inflammatory effects and release growth factors (e.g., VEGF, HGF) that can enhance wound healing and tissue regeneration.
The same platform is also being tested in the treatment of Crohn’s disease fistula, diabetic foot ulcer and burn injury.
Discussing the possibilities in combining gene therapy and cell therapy in RDEB treatment, Jakub Tolar and colleagues at University of Minnesota turned their collective gaze on induced pluripotent stem cells (iPSCs).
“iPSCs resemble embryonic stem cells in their capacity to differentiate into the hundreds of different specialized cell types found in the complex tissues of mammalian organs,” they wrote last year. “This broad differentiation profile and their use as disease modeling tools makes them uniquely suited for RDEB regenerative therapies.
“Specifically, the derivation of iPSCs from RDEB fibroblasts and keratinocytes, and the genetic correction of these cells—along with rigorous quality assurance and controls to remove the risk of reprogramming-based mutation accumulation—hold great promise for RDEB therapy.”
According to the authors, whether the next-generation therapies are gene- or cell-based and provided systemically or locally, they will involve a complex multistep process.
“To be useful in the clinic, these steps—including design, production and delivery of molecular tools; preparation and expansion of relevant cells; and local or systemic grafting of the gene-edited cells—must be validated and simplified,” they concluded (see also sidebar article “Beyond the incubator” below). “The common theme in successful examples of translation is a disappearance of the distance between the bench and the bedside with a cognizance that to make such research clinically meaningful: the technology has to be robust, scalable, suitable for regulatory approval and achieve more complete therapeutic outcomes.”
Beyond the world of life-threatening skin conditions, however, stem cells are also making headway in areas that were once largely the domain of late-night informercials: hair replacement and wrinkles.
Hair today, gone tomorrow
“Androgenic alopecia is the primary reason for baldness in men, and a significant reason for thinning hair in women; about 70 percent and 30 percent, respectively,” notes Lee Buckler, president, CEO and director of RepliCel Life Sciences.
From their research on hair follicles, company co-founders Rolf Hoffman (chief medical officer) and Kevin McElwee (chief scientific officer) wondered why the hair at the back of the head seemed to be “immune” to loss while those on the top of the head disappeared.
“We know from a couple of decades of hair transplant surgery that if we relocate those follicles and all the cells embedded in and around it to the affected area at the top, those cells will remain immune to the condition and continue to grow new hair fibers in that transplanted follicle, hair cycle after hair cycle, even though those cells are now probably depending on their progeny to do so,” Buckler explains.
What Hoffman and McElwee discovered was that the androgen hormone binds to a receptor on specific cells in the bulb of the hair follicle, causing them to die off and the hair fiber to disappear. In the hairs at the back of the head, however, those same cells—the dermal sheath cup cells—simply lack that receptor and so remain vital even in the presence of the androgen hormone.
“Rather than relocating thousands of follicles from the back of the head to the top of the head, which is highly invasive micro-transplant surgery, could we simply isolate that cell population and grow more of them in the lab and successfully repopulate the dormant hair follicles at the top of the head with a cell population that is immune to the condition?” Buckler asks.
The answer was a resounding yes.
“We started on mice, and we grew hair on paw pads, where there are no follicles at all,” Buckler recounts. “We grew whisker hair on ears in mice. We fluorescent tagged the cells and watched them migrate and take up residence in dormant hair follicles.
“And we eventually moved it into humans and saw some very nice responses in a Phase 1 human clinical study.”
Working with Shiseido, RepliCel’s lead product RCH-01 is currently undergoing clinical study at Tokyo Medical University Hospital and Toho University Ohasi Medical Center.
For Buckler, the collaboration with Shiseido is critical to his company’s success downstream.
“Our whole business model is built around putting it commercially into the hands of well-established commercial partners with experience in the clinical markets that our products are targeted for,” he says.
His day, as he describes it, is “to develop as much value in these assets as possible, get them on the radar screens of the right commercial partners and make sure that when the time is right, we’ve got commercial partners that are excited about partnering with us to complete late-stage, if necessary, and/or to launch these things and distribute them commercially.”
RepliCel and Shiseido are not alone in this approach to hair replacement, however. In 2016, materials specialist Kyocera announced its collaboration with RIKEN and Organ Technologies to tackle the alopecia market using stem cell technology.
As with RepliCel, the Japanese platform starts with a punch biopsy of skin from the back of the head, but they then isolate epithelial stem cells and follicle dermal papilla cells (mesenchymal stem cells), reconstituting the cells within a collagen gel to generate what the group calls a follicular primordium.
In preclinical studies to date, the group has transplanted such follicular primordia into hairless mice and demonstrated growth of new hair follicles. Furthermore, they suggest that the primordia connect to surrounding tissues such as nerves and arrector pili muscle, the muscles that responsible for goosebumps.
As well, the method allows for the addition of pigment stem cells, offering the potential for aesthetic treatment of alopecia.
Unlike stem cell efforts in most other therapeutic applications, however, efforts like those at RepliCel are based on not reinventing the cell but rather letting the cells do what they do best.
“We have a very simple philosophy of life in this area,” Buckler says. “I have no doubt that there will be tremendous successes down the road with all kinds of more complicated stem cell plays where you’re really relying on the stemness of these cells, differentiate them, modify them, inject them systemically for systemic conditions.”
RepliCel, however, was more interested in what they consider a lower-risk approach to cell therapy, identifying a cell population that plays a particular function and in terms of manufacturing those cells into a product, trying to maintain that function, and injecting them locally rather than systemically to have a local effect.
“We think that this represents a much lower risk approach,” he continues. “In terms of not having to introduce now complicated stem cell differentiation, we’re not looking at gene modification, we’re not looking at systemic injections or potential toxicities or requiring cells to hone to any particular locations.”
“So the science and the manufacturing is a lot more binary here, and the clinical impact we’re looking for is much more localized and measurable,” he presses, bluntly adding, “and there are significantly fewer areas for us to screw up.”
Hair follicle exploration also offered another opportunity to RepliCel researchers as they eventually characterized not only the dermal sheath cup cells, but also the cells within the tissues comprising or adjacent to the embedded hair follicles.
“While we were playing around with these cells, we discovered that the fibroblast cells, which are responsible for growing the hair follicle tissue, are extremely expressive of tissue-building proteins—more expressive than skin-derived fibroblasts—and particularly expressive of one of the proteins called type 1 collagen,” says Buckler.
“We went to clinical advisors and asked where is there a deficit of type 1 collagen that these cells might be useful in addressing?” he continues. “Skin immediately came to mind.”
According to the American Society for Plastic Surgeons, in 2013 alone, Americans spent $2.5 biliion on facial aesthetics, and this number was expected to grow to more than $5.4 billion by 2020, with dermal filler procedures accounting for more than 15 percent of those costs.
In April, RepliCel announced interim findings of its Phase 1 study evaluating its lead product RCS-01 in the treatment of aging and sun-damaged skin, relying on intradermal injections of hair-follicle-derived fibroblasts (or non-bulbar dermal sheath [NBDS] cells).
Although the study was not designed to look at efficacy, the researchers noted significant increases in the gene expression of several collagen-related biomarkers after just a single injection, including TIMP.
“This type of positive effect on TIMP gene expression, which is related to protection against collagen degeneration, is rarely observed,” offered Jean Krutmann, scientific manager of the IUF Leibniz Research Institute for Environmental Medicine. “In my experience, after decades of performing these tests, this is an exceptional finding, particularly for a safety trial with a small sample size.”
RepliCel’s Hoffman offered an additional benefit that “because RCS-01 is comprised of cells derived from tissue at the back of the patient’s scalp, these cells are not only very good collagen producers, but also UV-protected and therefore more functionally active.”
The active use of quantifiable biomarkers is an important facet of such studies, says Buckler.
“Dermatology is filled with what I call fuzzy endpoints, sort of the before-and-after pictures, in a race to get a product to market,” he explains. “We really wanted to understand if we injected these really highly collagen-expressive cells under the skin, were we changing the composition of the extracellular matrix (ECM) in a material way?”
With safety as a primary endpoint, the company wanted before-and-after biopsies for hard data that was less prone to subjective analysis.
“Some of the samples went first to pathology to look for any anomalies in the tissue,” Buckler continues. “Other samples went for PCR analysis looking for impact on 10 different biomarkers, which we established with our clinical advisors at the beginning of the trial, which were highly correlated in the published literature with aging and sun-damaged skin, on the premise that if we weren’t making any impact on those biomarkers, we didn’t have anything worth continuing to pursue.”
As suggested from Krutmann’s comments above, there was a clear impact of treatment.
Disease treatment isn’t the only potential application here, however.
Models showing skin
Increasingly, as stem cell technologies allow researchers to generate almost any type of cell, opportunities have opened to make more and more accurate models of skin itself. Rather than use this skin to cover wounds, however, researchers see increasing opportunities to use it as a platform to test the mechanisms, delivery and safety of new drugs, industrial chemicals and cosmetics.
“Human 3D skin equivalents (HSEs) are in-vitro models used to conduct experiments on processes involving the skin—e.g., disease progression and drug discovery,” wrote Columbia University’s Angela Christiano and colleagues in 2015. “Currently, however, the utility of HSEs is limited since these models lack the full cellular complexity of human skin; i.e., they contain few cell types and no appendages.”

One challenge, they explained, is that the models consist of cells isolated from freshly discarded tissue following surgery, which is typically quite small. Thus, it is often only the most abundant cells such as keratinocytes and fibroblasts that can be isolated and expanded in culture.
With the potential for unlimited growth and differentiation, iPSCs offer an alternative that also retains the genetic legacy of the donor tissue (e.g., gene deletions or mutations).
Years earlier, the group demonstrated the feasibility of generating HSEs from iPSC-derived fibroblasts and keratinocytes, whether in the study of normal skin or using tissues derived from RDEB patients.
“In [the 2015] study, our goal was to increase the complexity of these HSEs by adding additional iPSC-derived cell types, to better recapitulate normal human skin,” the authors wrote.
“Epidermal melanocytes represent the second most abundant cell type found in normal human epidermis and are largely responsible for both skin color and protection against the damaging effect of ultraviolet radiation via production of melanin,” they explained. “Moreover, pigmentation changes are often observed in response to disease or drug treatments, making HSEs that incorporate melanocytes an important tool in the drug discovery process.”
Combining iPSC-derived fibroblasts, keratinocytes and melanocytes, the researchers generated skin equivalents with normal tissue architecture. And most importantly, the melanocytes homed to the basal epidermis where they produced melanin.
A year later, Takashi Tsuji and colleagues at Tokyo University of Science, RIKEN and Organ Technologies—the group working with Kyocera on hair replacement—took skin modeling one step further, using iPSCs to not only generate skin, but also the appendage organs such as “hair follicles and sebaceous glands with proper connections to the surrounding tissues, such as the epithelium, dermis, fat, arrector pili muscles and nerve fibers.”
In this case, the researchers cultured iPSCs until they formed embryoid bodies, which were then transplanted into SCiD mice. The transplantation phase allowed the embryoid bodies to further differentiate into the cell types required to generate the skin as well as the appendages.
For its part, RepliCel is doing a molecular deep-dive, recently teaming up with researchers at University of British Columbia to generate a complete gene and protein expression profile of four of the different cell populations in the hair follicle.
“At the end of the day, it is driven very simply by our ardent belief that the more that we can understand about the cells, the product and the patient, the more intelligent decisions we can make about manufacturing, clinical trial design, efficacy, etc.,” says Buckler.
He proposes that if autologous cell therapy manufacturing is going to be commercially viable, its future lies with closed, highly automated manufacturing systems that can be located much closer to the patients.
“We’re looking to understand the particular genetic and protein expression profile of the cell population of interest and expect that this might lead us to isolate that cell population in a much more automated way; i.e., flow cytometry, filtration, some more high-throughput automated system,” he says.
Secondly, this information will eventually be important for any release assays to assure the FDA that any resulting product is fully characterized and sufficiently defined to be allowed onto the market.
“And then the third benefit is that we have now from our studies a pocket of responders that responded very nicely to treatment,” Buckler finishes. “We have an opportunity to understand better if we can correlate a particular genetic or protein expression profile to clinical efficacy.”
Of course, the five men in London will likely remain completely oblivious to all of this, just like the millions of people around the world who struggle with wounds and dermatological diseases. For them, this is all just background noise—however vital to their future well-being—as they push forward with their lives, forever changed.

Beyond the incubator
Despite decades of technological advances and clinical promise, there is still a significant commercial lag for stem cell-based therapies—or any cell or gene therapies, for that matter—compared to their small-molecule and biologics brethren. In part, this is due to the rather large hurdles of converting something that shows promise in multiwell plates and the clinic into something that delivers in the less-controlled environs of hospitals.
“One of the shortcomings of some management teams I’ve seen in the past is the assumption that if they have a great product and they get it approved, everything else will solve itself,” explains RepliCel Life Sciences President, CEO and Director Lee Buckler. “And I think nothing could be farther from the truth.”
Although having a working product that gets approved by the FDA may not be the beginning of the battle, he admits, those steps remain only part of the battle.
“What we really have to focus on are products that make commercial clinical sense,” he presses. “Sometimes a product that works doesn’t make sense.”
Buckler suggests that because there are so few examples of companies that have managed to achieve commercial success with autologous cell therapies, the entire field remains somewhat nervous as it moves forward.
With that in mind, RepliCel determined to learn what it could from two examples that it really felt typified the hazards: Dendreon and Fibrocell.
“One of the challenges of the Dendreon experience is that they over-built in terms of their manufacturing capacity when it came to commercial launch,” Buckler explains. “I used to work for a contract manufacturer and certainly not only because of that, but also because of the experience I have seen with people over-building bricks and mortar too early, makes me rely on keeping things very virtual.”
Another challenge, he continues, was that every time they wanted to do an injection—and this was a three-injection protocol—they had to start all the way from scratch.
“So, they had to collect, manufacture, do their final release and then deliver the product, and it had to be injected within, I think, 72 hours of release from the manufacturing facility,” he describes. “And maybe it was impossible for them, but one of the reasons why that was so was because they didn’t cryopreserve the product.”
Says Buckler, this meant that Dendreon had to spend a lot of resources coordinating the patient and the doctor’s availability with the release of the product from the manufacturing facility, because if it didn’t get injected, it was dead and then they had to start all the way from scratch again.
Learning from that lesson, RepliCel invested very early on in cryopreservation such that, even though their product is autologous, they can scale to patient needs, which gives them a lot more logistical freedom.
Like RepliCel, Fibrocell works in the dermatology space, using gene modification in autologous fibroblasts for the treatment of deep wrinkles, as well as several other projects.
“What we’ve all learned from the Fibrocell case study are a couple of things,” Buckler says. “One is that they hadn’t continued to invest in manufacturing optimization, and so, by the time they got to approval, their manufacturing looked quite antiquated to current state of the art. It took very long to manufacture that product.”
“But even more importantly for our purposes was what happened after commercial launch,” he presses.
Although the Fibrocell product had solid efficacy throughout the clinical phase of development, efficacy that got them through a BLA approval from the FDA, Buckler offers, these outcomes became less certain once the product moved out of the controlled environment of clinical studies and into the hands of clinicians who might perform one or two injections a month.
“What Fibrocell has attributed this to is that there was too much variability in the injection process,” he suggests. “They were really relying on training sessions and weekends that they would put these clinicians through and expecting them to be consistent with the treatment and the injection protocols and the practices of the primary investigators in the clinical trials.”
Again, learning the lessons of their predecessors and competitors, RepliCel became “irrationally committed,” as Buckler describes it, to the co-development of an injector that dictated absolute precision and therefore consistency of cell injection.
“It sounds trite to say now, but we’re committed to a very simple rationale that is if you’re not controlling the injection process, you’re not controlling the outcome,” he says.
It ultimately comes down to protecting the company and its stakeholders’ investment.
“We could have the best biologic in the world, but if someone kills it on the way in or injects it sloppily, it’s going to flop. That’s no good for anybody.”

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