Several new treatments for patients with limbal stem cell deficiency have emerged in the last two decades. These cell-based therapies use smaller amounts of donor tissue compared to standard approaches like conjunctival limbal autografts and many also involve expanding cultured cells in the lab. Here, I’ll discuss these approaches and share the latest results of a new stem cell therapy for cornea.

 

Limbal Stem Cells’ Form and Function

Corneal limbal stem cells are specialized adult stem cells that reside in the corneal periphery. These cells carry out several functions and are essential for maintaining the integrity and transparency of the cornea. They serve as the source of corneal epithelial cells, continuously regenerating the epithelial layer, and act as a barrier to prevent the conjunctiva from migrating onto the corneal surface. When limbal stem cells function properly, they ensure the cornea remains clear and free of vascularization.

Limbal stem cell deficiency occurs when these corneal cells are damaged or lost due to factors such as trauma, chemical burns, infections and immunologic or genetic disorders. As a result, the corneal epithelium can’t regenerate properly, leading to pannus and conjunctivalization of the cornea. This process results in vision loss, blindness and significant pain.

 

Existing Treatments for LSCD

Chemical injury resulted in complete loss of limbal stem cells (top). Photos of the same eye two weeks after CALEC transplantation (middle) and 18 months after the procedure (bottom).

In the early stages of LSCD, conservative medical treatments can often provide relief. These include the use of artificial tears, punctal plugs, anti-inflammatory eye drops and serum tears. In more advanced stages, amniotic membrane transplantation may be used as a scaffold to support the growth of remaining limbal stem cells. However, when LSCD becomes severe and the stem cell reserve in the peripheral cornea is exhausted, surgical intervention is required to replace the lost or damaged stem cells.

Current standard treatment for unilateral disease is a conjunctival limbal autograft, which involves harvesting limbal tissue from the patient’s healthy fellow eye. Typically, this involves taking up to four clock hours of limbal tissue and transplanting it onto the diseased eye. This method is considered safe and effective at replenishing the stem cell population, but it also carries significant risks, including damage to the donor site and the potential for providing an insufficient barrier for preventing the recurrence of the limbal stem cell deficiency.

 

Innovative Techniques

Newer autologous epithelial transplantation techniques such as cultivated limbal epithelial transplantation (CLET) and simple limbal epithelial transplantation (SLET) have been developed as alternatives to CLAU. These methods aim to reduce the risk of fellow eye damage by harvesting smaller amounts of limbal tissue. Both have gained widespread popularity among corneal transplant surgeons. SLET in particular is a cost-effective, straightforward procedure and doesn’t require the use of a lab for cell expansion.

Both of these approaches have demonstrated favorable long-term surgical outcomes, according to a literature review study of 103 eyes.1 CLET, SLET and COMET—cultivated oral mucosal epithelial transplantation—had success rates of 45.5 percent, 77.8 percent and 57.8 percent, respectively. Seven-year survival rates after each procedure were 50 percent, 72.2 percent and 53.2 percent, respectively. Patients with SJS/TEN had significantly lower survival rates across the board but these were significantly better after SLET.

 

Cultivated Limbal Epithelial Transplantation (CLET)

Autologous CLET opened the door for exciting new cell-based therapeutic avenues. In this limbal stem cell transplantation approach, a 1- to 2-mm2 portion of healthy fellow limbus is harvested and cells are expanded into cell sheets in vitro.2 Repeat procedures are possible due to the small biopsy size. This approach is suitable for eyes with severe unilateral or non-total bilateral LSCD. 

CLET has been found to provide a good foundation for subsequent penetrating keratoplasty.3 Additionally, allogeneic CLET has shown good safety and efficacy, though it carries the risk of rejection and systemic immunosuppression is needed.4

Previously, CLET’s cultivation and manufacturing processes involved the use of xenogeneic murine feeder cells and fetal bovine serum,5 and these are subject to strict regulation in the United States. As such, the procedure used to manufacture CLET doesn’t meet the FDA’s rigorous standards. Nevertheless, there’s ongoing research into stem cell expansion without xenobiotic components under Good Manufacturing Practice (GMP)-compliant conditions. Two clinical trials (NCT03957954 and NCT02592330) are underway for studying cultivated autologous limbal stem cells for LSCD. Induced pluripotent stem cells are also being investigated in Japan as a source of limbal epithelial cells (JPRN-UMIN000036539), as are mesenchymal stem cells (NCT01562002). 

CLET products are approved in other countries. In the EU, it’s marketed as Holoclar (Holostem Terapie Avanzate [Holostem Advanced Therapies] and Chiesi Farmaceutici S.p.A) and as Nepic in Japan (Japan Tissue Engineering Co., Ltd.). 

Holoclar’s Phase IV trial HOLOCORE is a multinational, interventional, prospective clinical study aimed at demonstrating the efficacy of autologous cultivated limbal stem cell transplantation.6 The study included 73 patients with moderate to severe LSCD due to ocular burns. Seven repeat procedures were performed. Holoclar demonstrated an acceptable success rate (41 percent; 25 out of 61 patients) at 12 months post-treatment, with a stable corneal surface and no surface epithelial defects (82 percent). Fifty-seven percent of patients saw a reduction in ingrown blood vessels; 75.4 percent reported no burning and 78.7 percent reported no pain at one year after the procedure. No signs of increased limbal blood were observed in approximately half of patients, and 37.7 percent had normal corneal sensitivity. Side effects included eye pain from corneal epithelial defect and corneal thinning, which led to three patients discontinuing the trial. Overall, the treatment was deemed successful at restoring the corneal surface in moderate to severe LSCD with symptom and vision improvement.

For Nepic, a prospective trial to confirm the efficacy and safety of GMP-compliant cell sheets reported successful corneal epithelial reconstruction in 60 percent of eyes at one year. This finding was significantly higher than the 15-percent clinically significant efficacy rate of allogenic limbal transplantation. At two years, the reconstruction rate was 70 percent, and visual acuity improvements were noted in 60 percent of eyes at two years. A retrospective study of five LSCD cases reported promising short- and medium-term results.7

 

Simple Limbal Epithelial Transplantation (SLET)

SLET was first described in 2012 as an approach aimed at improving upon both CLAU and CLET. Conceived for unilateral LSCD, it’s best suited for treating LSCD caused by situations such as chemical injury or medication toxicity. Good outcomes in conjunction with penetrating keratoplasty and DALK have been reported in the literature.8,9

In SLET, a small biopsy of limbal tissue (about one clock hour) is harvested from the healthy fellow eye and divided into several pieces. These pieces are distributed over an amniotic membrane, which is then placed over the diseased cornea. Complete in vivo re-epithelialization typically occurs within two weeks. SLET can be performed as a same-day procedure. 

This approach has been touted for its reduced risk of iatrogenic LSCD in the healthy fellow eyes due to the small biopsy size and its single-step approach that obviates laboratory cell expansion, which is costly and takes time. According to a literature review and online ophthalmic surgeon survey on SLET uptake,10 SLET has an anatomical success rate in adults of 72.6 percent and 77.8 percent in children, similar to CLET (70.4 percent in adults and 44.5 percent in children). Ninety-nine surgeons reported performing SLET on 1,174 patients. They like that SLET doesn’t require expensive tissue engineering facilities. The researchers’ economic analysis showed that SLET provides an estimated cost savings of $6,470.88 for adults and $6,673.10 for children. They added that SLET costs about 10 percent and 8 percent of CLET’s costs for adults and children, respectively.

Several other studies have reported comparable success rates for SLET compared with CLAU and CLET. A review of 31 SLET studies identified complete success in 83 percent of cases and visual acuity improvements in 69 percent of cases.11 Another study comparing CLET and SLET in 100 patients with unilateral LSCD due to ocular burns reported that SLET was equally effective in restoring ocular surface stability and provided better visual outcomes than CLET throughout three years of follow-up.12 

SLET was also found to be an effective CLET alternative in cases of recurrent LSCD after previous failed CLET.13 The authors suggest that SLET is preferable to repeat CLET since it’s a single-stage procedure and it’s less expensive.

Allogeneic SLET has also shown excellent efficacy. A study comparing autologous SLET (n=11) and living relative allogeneic SLET (n=17) in 28 eyes of 26 patients reported similar overall survival rates of 89.3 percent and 75.6 percent, respectively.14

Currently, SLET isn’t FDA approved. 

 

Cultivated Oral Mucosal Epithelial Transplantation (COMET)

The first use of cultured non-limbal autologous cells to treat bilateral LSCD was in 2004 with a COMET procedure. This approach using oral mucosa minimizes the risk of graft rejection by using autologous cells, but angiogenesis following transplantation is a key drawback. Favorable outcomes have been reported for a number of conditions, from chemical burns to SJS15 and chronic cicatrizing diseases.16 A 43- to 67-percent success rate has also been reported for restoring ocular surface stability in LSCD with COMET.17 

COMET launched in Japan as the product Ocural (Japan Tissue Engineering Co., Ltd.) in 2021. Early clinical findings of this product in two cases showed successful engraftment and that the procedure could be performed without major complications.18 Markers for stem cells p63 and p75, proliferation (Ki-67) and differentiation (Keratin-3, -4 and -13) were found in the cornea-like tissue after COMET and in the cultivated oral mucosal epithelial cell sheet.

CALEC’s Current Study Results

Sezen Karakus, MD, an assistant professor of ophthalmology and cornea specialist at Johns Hopkins, says that the early findings reported in the Phase I/II clinical trial for CALEC transplantation are promising. “The study demonstrated excellent feasibility and safety, with 93 percent manufacturing success (14 out of 15 participants received grafts that met all product release criteria),” she says. “Notably, in most cases, a single limbal biopsy was sufficient to generate two constructs, minimizing the need for repeat procedures.

“The clinical success rate, defined as improvement in corneal epithelial integrity, neovascularization and/or symptomatology (OSDI and SANDE), was high: Eighty-six percent at three months, 93 percent at 12 months, and 92 percent at 18 months achieved either complete or partial success,” she continues. “Only three participants required a second transplant, and these were cases with severe underlying or coexisting ocular conditions.

“Importantly, there were no serious safety concerns, including in the donor eyes from which biopsies were taken,” she notes. “There were no cases of donor site limbal stem cell deficiency. The only reported primary safety event was a single infection, unrelated to the graft and attributed to contact lens use.”

Dr. Karakus points out that while CALEC shows strong potential for future adoption, its broader clinical use in the United States may be limited by the need for GMP-compliant manufacturing and a highly controlled cell processing environment. “Since the graft must be used within 24 hours of final processing and can’t currently be cryopreserved, implementation may remain restricted to specialized centers,” she says.

As for where this new cell therapy might fit into the limbal stem cell deficiency treatment algorithm with existing procedures such as CLAU and SLET, Dr. Karakus says that CALEC may offer several compelling features that corneal specialists might appreciate. 

“Compared to CLAU, CALEC is less invasive, requiring a much smaller limbal biopsy, thereby reducing the risk of inducing LSCD in the healthy eye,” she says. “Even in the event of an initial culture failure, a second biopsy can be performed, and the total amount of harvested tissue would still likely be less than that required for CLAU. This tissue-sparing feature is particularly advantageous when donor eye preservation is a concern.

“Compared to SLET, CALEC offers theoretical advantages in terms of cell dose and quality,” she continues. “In SLET, the limbal tissue is placed directly on the recipient’s eye without prior expansion, relying on in vivo proliferation. CALEC, on the other hand, allows ex vivo expansion from the same biopsy size, likely yielding a higher number of functional stem cells, potentially translating to more robust and durable clinical outcomes. However, there’s no head-to-head clinical trial data comparing CALEC and SLET directly.

“That said, SLET remains a strong competitor, particularly in settings without access to specialized labs,” she explains. “It’s a technically simpler, cost-effective and widely accessible procedure that only requires an amniotic membrane and basic surgical facilities. However, neither SLET nor CALEC is currently FDA approved, although CALEC, with its compliance to FDA manufacturing standards, appears to be well-positioned to achieve regulatory approval in the United States in the future.”

She adds that CALEC’s major distinction lies in its compliance with FDA regulatory requirements, making it a frontrunner among cultivated cell therapies for potential approval in the United States. “While broader clinical adoption may be limited by the need for specialized manufacturing infrastructure, CALEC offers a promising, scalable and reproducible approach—particularly well-suited to tertiary care centers and complex LSCD cases where maximizing donor tissue preservation and product quality are critical,” she says.

 

CALEC manufacturing schema. In this approach, an approximately 2-mm section of limbal tissue is harvested from the healthy fellow eye. The stem cells then undergo expansion in the laboratory and are cultured into cell sheets. The cell sheet is transplanted onto the diseased eye.

CALEC Therapy

Like SLET and CLET, cultivated autologous limbal epithelial cell (CALEC) transplantation offers a less invasive alternative to CLAU since it involves taking only one clock hour, or about 2 mm, of limbal tissue from the fellow eye. This is followed by the expansion of stem cells in the laboratory. The cells are cultured into cell sheets and transplanted onto the diseased eye, offering a higher yield of cells compared to the traditional method. 

CALEC is the result of a collaborative effort among Mass Eye and Ear, Harvard Medical School, Dana Farber, Children’s Hospitals, the GMP laboratory and the National Eye Institute. 

A preclinical study19 sponsored by the National Heart, Lung and Blood Institute tested a two-step manufacturing process for limbal stem cell isolation and expansion. The Phase I trial enrolled five patients with unilateral LSCD, four of which received CALEC transplants successfully with no primary safety events. 

This past March, the results of the NIH/NEI-funded Phase I/II trial (n=14 participants) were published,20 demonstrating a 92-percent success rate at 18 months of follow-up, with improved corneal epithelial surface integrity and improvements in corneal vascularization and patient symptoms. Currently, there are no Phase III trials in the works due to funding constraints.

Before placing a new CALEC graft, the diseased epithelium is removed.

Here’s how this new procedure works:

First, the patient is brought to the operating room, where topical anesthesia. A biopsy is then harvested from the healthy eye. It’s crucial to ensure that the limbus appears healthy, with a clear Palisades of Vogt and minimal exposure to ultraviolet light. For optimal tissue quality, I prefer to collect biopsies from the superior corneal area.

The biopsy typically measures approximately 2 mm. During the limbal biopsy, the conjunctiva is usually excised as well to prevent cross-contamination from conjunctival cells. The harvested tissue is then placed in an epithelial transport medium, a solution we’ve developed specifically for this purpose. 

The tissue is transported to a GMP-certified laboratory, which holds special accreditations for producing human-grade products such as gene therapy or bone marrow transplantation. The GMP laboratory has a highly coordinated protocol for extracting and processing the cells from the biopsy to ensure optimal quality and compliance with stringent regulatory standards.

The limbal biopsy is then subjected to enzymatic digestion, where enzymes break down the tissue, and the epithelial cells are manually scraped from the remaining tissue. These cells are carefully placed on a plastic culture plate. Our medium is then added to support the growth of the cells. Once a confluent monolayer of epithelial cells has formed, a series of quality control assays are performed on a portion of these cells. These tests include microbiological evaluations, assessments of stem cell markers and checks for cross-contamination by non-epithelial cells. Only when the cells pass these tests are they deemed suitable for the next step.

The cells are then transferred to a secondary culture stage, where they are placed on a de-epithelialized amniotic membrane. This membrane serves as a scaffold for the cells and forms the basis for the final graft. This step typically takes approximately one week. Throughout this time, additional testing is conducted, including imaging of the cells using an EVOS system, which functions similarly to a specular microscope, allowing for detailed observation of the cellular morphology and confluence.

A CALEC graft is placed on the eye and maneuvered into place over the cornea before it’s secured with sutures. (View a video of the procedure below).

Once the graft reaches the predetermined quality standards—based on our established parameters—it’s ready for transplantation onto the patient.

At this stage, the patient typically presents with significant ocular pathology due to limbal stem cell deficiency. A retrobulbar block is performed to provide anesthesia, ensuring that the eye is numbed effectively. The surgical procedure involves meticulous dissection to remove scar tissue and pannus, with special attention to controlling bleeding. In such cases, I often use epinephrine to constrict blood vessels and minimize blood loss. The limbal pannus is carefully excised, and any symblepharon are lysed. Reconstruction of other areas may be necessary before proceeding with the placement of the CALEC graft.

The CALEC graft is then positioned onto the prepared ocular surface and sutured into place with 10-0 nylon sutures. To further shield and protect the newly grafted cells, a contact lens is placed on the eye. 

One limitation of CALEC therapy is that it’s currently applicable only to unilateral cases. However, as we know, limbal stem cell deficiency often affects both eyes bilaterally. While this remains a significant hurdle, it’s an important first step in demonstrating the feasibility of cell-based therapies for corneal restoration. This represents, to our knowledge, the first stem cell therapy specifically for the cornea in the United States.

Moving forward, a critical challenge will be developing strategies for allogeneic transplantation. Fortunately, much of the groundwork has already been laid. Our protocol development involved using donor corneas, where small biopsies were taken from cadaveric tissue and cultured prior to transitioning to patient corneas. These preclinical trial runs have allowed us to refine our techniques and confirm their effectiveness. Now, we’ll return to these trial models and adapt our protocol for the use of allogeneic tissue in the clinical setting.



Dr. Jurkunas is a professor of ophthalmology at Harvard Medical School and a clinician-scientist at Massachusetts Eye and Ear, where she’s also the associate director of the Cornea Service. 

CALEC is patent pending. Dr. Jurkunas has a financial interest in OcuCell, which is slated to license the cell therapy. OcuCell was not involved in the trials discussed in this article and no licenses have been granted as of this writing.

 

 

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