Cellular therapy of the corneal stroma: Real approach or science fiction?

Corneal transplantation has experienced great evolution in the past decade, thanks to the advancements in endothelial transplantation techniques. However, corneal stroma rehabilitation is still limited by the drawbacks of penetrating (PK) and deep anterior lamellar keratoplasty (DALK) techniques: slow visual recovery, rejection risk for PK, shortage of donor tissue and long learning curve for DALK.

Despite the great efforts made in recent years to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation, this has not yet been accomplished – due largely to the extreme difficulty in mimicing the highly complex ultrastructure of the corneal stroma; obtaining substitutes with either insufficient transparency or insufficient strength.

Cellular therapy of the corneal stroma is currently being investigated as a possible treatment option to alleviate corneal stroma opacities. Extraocular mesenchymal stem cells (those derived from the adult adipose tissue) are an ideal source of autologous stem cells, since the tissue is easily accessible, there is high cell retrieval efficiency, and the cells are able to differentiate in multiple cell types (keratocytes, osteoblasts, chondroblasts, myoblasts, hepatocytes, neurons, etc).¹

This has been demonstrated in animal models, as recorded by several authors, including reports from our own research group.^2-4 We have seen that ocular and extraocular mesenchymal stem cells are able not only survive to and differentiate into adult human keratocytes in xenogeneic scenarios without inducing any inflammatory reaction, but also to produce new collagen within the host stroma.^2,5

The cells can also modulate pre-existing scars by corneal stroma remodelling^6,7 and improve corneal transparency in animal models for corneal dystrophies by collagen reorganisation as well as in animal models for metabolopathies by the catabolism of the accumulated proteins.^8-11

Mesenchymal stem cells have also shown immunomodulatory properties in syngeneic, allogeneic and even xenogeneic scenarios.^11,12

Advanced keratoconus study

Our group has conducted and recently published the first human clinical trial designed to preliminarily evaluate the safety and efficacy of the cellular therapy with mesenchymal stem cells for the corneal stroma.¹³ We performed a non-randomised and unblind clinical trial [ClinicalTrials.gov; Identifier: NCT02932852], which enrolled patients with advanced keratoconus (stage ≥ IV according to the RETICS keratoconus classification¹⁴) who were already candidates for corneal transplantation for their visual rehabilitation.

Five consecutive patients were included, and a suspension containing autologous adipose-derived adult stem cells (ADASC) was transferred into a mid-stromal femtosecond laser-assisted lamellar pocket. All procedures were conducted under topical anaesthesia and without requiring any corneal suture.

Fat containing the stem cells had previously been obtained by elective liposuction (under local anaesthesia) and subsequently processed in the laboratory in order to isolate the autologous ADASC (similar to the methodology reported in our previous experimental studies,^2-4 although a certified white chamber was required for the stem cells’ isolation this time, given their human application). (Figure 1.)

There were no intra- or postoperative complications. Corneal transparency was fully recovered to preoperative levels within 48 hours, and any inflammatory reaction or rejection response was documented in the 6-month follow-up to the study (Figure 2).

The mean age of the study patients was 34.2 years and none of them had previously received any ophthalmic surgical intervention. All patients had improved visual function: unaided and corrected distance visual acuities presented an overall improvement of more than one line compared with preoperatively, while best visual acuity with rigid contact lenses showed a total mean improvement of more than two lines.

Conversely, all keratometric parameters presented relative stability. A mild mean improvement of 16.5 µm in the central corneal thickness was detected by anterior segment OCT (Visante) at the sixth month, correlating with the demonstration of newly formed collagen in the majority of the patients – seen as patched hyperreflective areas at the level of the stromal pocket not homogeneously distributed along it (Figure 3).

Implanted ADASC survival was confirmed by confocal biomicroscopy (HRT-3, Heidelberg), where rounded-shape cells were observed in the surgical plane in all cases up to the third postoperative month (Figure 4). By the sixth postoperative month, these cells presented a fusiform shape and were not different from those observed in other stromal planes, assuming this morphologic change as a possible sign of adult keratocyte differentiation of the ADASC.

An interesting finding was a moderate clinical improvement of preoperative anterior stromal scars in one patient along the follow-up. This finding would agree with Dr James Funderburgh’s and other authors’ findings in animal models regarding the potential capability of mesenchymal stem cells to alleviate pre-existing corneal stromal scars.^6-9

Discussion points

To the best of our knowledge, this is the first paper reporting the clinical use of stem cells for the corneal stroma.¹³ Nevertheless, our results should be considered carefully given that this pilot clinical trial is vulnerable to many biases: i.e., small study sample and the unrandomised, unblind trial design. However, we have observed encouraging results that will open a new and very exciting line of research in the coming years.

All patients gained some vision (although not enough to consider it a viable alternative to classical corneal transplantation), and in our small study sample the implantation of autologous mesenchymal stem cells have  proven completely safe.

Probably one of the most important findings of this study is that a mild amount of newly formed collagen was generated within the stroma without the induction of any clinically visible stromal haze.

Of course, the study raises many questions regarding this novel therapy: are these findings clinically relevant and can they be enhanced by alternative approaches? Is the therapy able to halt the natural progression of the disease in less advanced cases?

All of these questions will have to be answered by future studies in order to establish whether this new modality of treatment can be accepted in real clinical practice, otherwise it will remain a scientific curiosity.

References

1.     De Miguel MP, Casaroli-Marano RP, Nieto-Nicolau N, et al. Frontiers in Regenerative Medicine for Cornea and Ocular Surface. In: Rahman A, Anjum S. Frontiers in Stem Cell and Regenerative Medicine Research. 1st ed. PA: Bentham e-Books, Vol. 1, 2015;92-138.

2.     Arnalich-Montiel F, Pastor S, Blazquez-Martinez A, et al. Adipose-derived stem cells are a source for cell therapy of the corneal stroma. Stem Cells. 2008;26:570–579.

3.     Alió del Barrio JL, Chiesa M, Gallego Ferrer G, et al. Biointegration of corneal macroporous membranes based on poly(ethyl acrylate) copolymers in an experimental animal model. J Biomed Mater Res A. 2015;103:1106–1118.

4.     Alió del Barrio JL, Chiesa M, Garagorri N, et al. Acellular human corneal matrix sheets seeded with human adipose-derived mesenchymal stem cells integrate functionally in an experimental animal model. Exp Eye Res. 2015;132:91–100.

5.     Espandar L, Bunnell B, Wang GY, et al. Adipose-derived stem cells on hyaluronic acid-derived scaffold: a new horizon in bioengineered cornea. Arch Ophthalmol. 2012;130:202–208.

6.     Mittal SK, Omoto M, Amouzegar A, et al. Restoration of Corneal Transparency by Mesenchymal Stem Cells. Stem Cell Reports. 2016;7:583-590.

7.     Demirayak B, Yüksel N, Çelik OS, et al. Effect of bone marrow and adipose tissue-derived mesenchymal stem cells on the natural course of corneal scarring after penetrating injury. Exp Eye Res. 2016;151:227-235.

8.     Du Y, Carlson EC, Funderburgh ML, et al. Stem cell therapy restores transparency to defective murine corneas. Stem Cells. 2009;27:1635–1642.

9.     Liu H, Zhang J, Liu CY, et al. Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice. PLoS One. 2010;5:e10707.

10.  Coulson-Thomas VJ, Caterson B, Kao WW. Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice. Stem Cells. 2013;31:2116–2126.

11.  Kao WW, Coulson-Thomas VJ. Cell Therapy of Corneal Diseases. Cornea. 2016;35 Suppl 1:S9-S19.

12.  De Miguel MP, Fuentes-Julián S, Blázquez-Martínez A, et al. Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr Mol Med. 2012;12:574-591.

13.  Alió del Barrio JL, El Zarif M, de Miguel MP, et al. Cellular Therapy with Human Autologous Adipose-derived Adult Stem Cells for Advanced Keratoconus. Cornea. 2017;36:952-960.

14.  Alió JL, Piñero DP, Alesón A, et al. Keratoconus-integrated characterization considering anterior corneal aberrations, internal astigmatism, and corneal biomechanics. J Cataract Refract Surg. 2011;37:552-568.

Dr Jorge L. Alió del Barrio, MD, PhD

E: jorge_alio@hotmail.com

Dr Alió del Barrio is a surgeon at the Cornea, Cataract and Refractive Surgery Unit, Vissum Corporación, Alicante, Spain and professor “ad Honorem” at the Miguel Hernández University’s School of Medicine, Alicante, Spain. He has no financial interests to disclose.

Professor Jorge L. Alió MD, PhD, FEBOphth

E: jlalio@vissum.com

Prof. Jorge L. Alió is professor and chairman of ophthalmology at the University Miguel Hernández de Elche, Alicante, Spain. He is also founder of Vissum Corporation, Spain, and of the Jorge Alió Foundation for the Prevention of Blindness. He has no financial interests in the subject matter.

Dr Mona El Zarif, OD

E:monazarifaj@hotmail.com

Dr El Zarif is the owner of Optica General S.A.R.L. Saida in Lebanon and head of the Optometry and Contactology department. She has no financial disclosures to make.