UK Keratoconus Self-Help and Support Association

International Journal of Medical Sciences
2019; 16(4): 507-512. doi: 10.7150/ijms.30759
Review
Engineering of Human Corneal Endothelial Cells In Vitro
Qin Zhu1, Yingting Zhu2, Sean Tighe2, Yongsong Liu3 and Min Hu1
1. Department of Ophthalmology, The Second People's Hospital of Yunnan Province (Fourth Affiliated Hospital of Kunming Medical University); Yunnan Eye
Institute; Key Laboratory of Yunnan Province for the Prevention and Treatment of ophthalmology (2017DG008); Provincial Innovation Team for Cataract
and Ocular Fundus Disease (2017HC010); Expert Workstation of Yao Ke (2017IC064), Kunming, 650021 China
2. Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, 33173 USA
3. Department of Ophthalmology, Yan' An Hospital of Kunming City, Kunming, 650051, China
 Corresponding authors: Min Hu, M.D., Ph.D. Department of Ophthalmology, Fourth Affiliated Hospital of Kunming Medical University, Second People's
Hospital of Yunnan Province, Kunming 650021, China; Telephone: 0118615087162600; Fax: 011860871-65156650; E-mail: fudanhumin@sina.com Or Yingting
Zhu, Ph.D. TissueTech, Inc., 7000 SW 97th Avenue, Suite 212, Miami, FL 33173. Telephone: (786) 456-7632; Fax: (305) 274-1297; E-mail: yzhu@tissuetechinc.com

Received: 2018.10.17; Accepted: 2019.01.10; Published: 2019.03.10
Abstract
Human corneal endothelial cells are responsible for controlling corneal transparency, however they
are notorious for their limited proliferative capability. Thus, damage to these cells may cause
irreversible blindness. Currently, the only way to cure blindness caused by corneal endothelial
dysfunction is via corneal transplantation of a cadaver donor cornea with healthy corneal
endothelium. Due to severe shortage of donor corneas worldwide, it has become paramount to
develop human corneal endothelial grafts in vitro that can subsequently be transplanted in humans.
Recently, we have reported effective expansion of human corneal endothelial cells by
reprogramming the cells into progenitor status through use of p120-Kaiso siRNA knockdown. This
new reprogramming approach circumvents the need of using induced pluripotent stem cells or
embryonic stem cells. Successful promotion of this technology will encourage scientists to re-think
how "contact inhibition" can safely be perturbed to our benefit, i.e., effective engineering of an in
vivo-like tissue while successful maintaining the normal phenotype. In this review, we present
current advances in reprogramming corneal endothelial cells in vitro, detail the methods to successful
engineer human corneal endothelial grafts, and discuss their future clinical applications to cure
corneal blindness.
Key words: cornea, endothelium, progenitor, engineering

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535652/

Review Exp Eye Res. 2012 Feb;95(1):16-23. doi: 10.1016/j.exer.2011.08.014. Epub 2011 Aug 30.

Proliferative capacity of corneal endothelial cells

Nancy C Joyce 1
Affiliations expand
PMID: 21906590 PMCID: PMC3261346 DOI: 10.1016/j.exer.2011.08.014
Free PMC article
Abstract
The corneal endothelial monolayer helps maintain corneal transparency through its barrier and ionic "pump" functions. This transparency function can become compromised, resulting in a critical loss in endothelial cell density (ECD), corneal edema, bullous keratopathy, and loss of visual acuity. Although penetrating keratoplasty and various forms of endothelial keratoplasty are capable of restoring corneal clarity, they can also have complications requiring re-grafting or other treatments. With the increasing worldwide shortage of donor corneas to be used for keratoplasty, there is a greater need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. As a result, researchers have been exploring alternative approaches that could result in the in vivo induction of transient corneal endothelial cell division or the in vitro expansion of healthy endothelial cells for corneal bioengineering as treatments to increase ECD and restore visual acuity. This review presents current information regarding the ability of human corneal endothelial cells (HCEC) to divide as a basis for the development of new therapies. Information will be presented on the positive and negative regulation of the cell cycle as background for the studies to be discussed. Results of studies exploring the proliferative capacity of HCEC will be presented and specific conditions that affect the ability of HCEC to divide will be discussed. Methods that have been tested to induce transient proliferation of HCEC will also be presented. This review will discuss the effect of donor age and endothelial topography on relative proliferative capacity of HCEC, as well as explore the role of nuclear oxidative DNA damage in decreasing the relative proliferative capacity of HCEC. Finally, potential new research directions will be discussed that could take advantage of and/or improve the proliferative capacity of these physiologically important cells in order to develop new treatments to restore corneal clarity.

https://pubmed.ncbi.nlm.nih.gov/21906590/

Clinical Trial N Engl J Med. 2018 Mar 15;378(11):995-1003. doi: 10.1056/NEJMoa1712770.

Injection of Cultured Cells with a ROCK Inhibitor for Bullous Keratopathy

Shigeru Kinoshita 1, Noriko Koizumi 1, Morio Ueno 1, Naoki Okumura 1, Kojiro Imai 1, Hiroshi Tanaka 1, Yuji Yamamoto 1, Takahiro Nakamura 1, Tsutomu Inatomi 1, John Bush 1, Munetoyo Toda 1, Michio Hagiya 1, Isao Yokota 1, Satoshi Teramukai 1, Chie Sotozono 1, Junji Hamuro 1
Affiliations expand
PMID: 29539291 DOI: 10.1056/NEJMoa1712770
Abstract
Background: Corneal endothelial cell (CEC) disorders, such as Fuchs's endothelial corneal dystrophy, induce abnormal corneal hydration and result in corneal haziness and vision loss known as bullous keratopathy. We investigated whether injection of cultured human CECs supplemented with a rho-associated protein kinase (ROCK) inhibitor into the anterior chamber could increase CEC density.

Methods: We performed an uncontrolled, single-group study involving 11 persons who had received a diagnosis of bullous keratopathy and had no detectable CECs. Human CECs were cultured from a donor cornea; a total of 1×106 passaged cells were supplemented with a ROCK inhibitor (final volume, 300 μl) and injected into the anterior chamber of the eye that was selected for treatment. After the procedure, patients were placed in a prone position for 3 hours. The primary outcome was restoration of corneal transparency, with a CEC density of more than 500 cells per square millimeter at the central cornea at 24 weeks after cell injection. Secondary outcomes were a corneal thickness of less than 630 μm and an improvement in best corrected visual acuity equivalent to two lines or more on a Landolt C eye chart at 24 weeks after cell injection.

Results: At 24 weeks after cell injection, we recorded a CEC density of more than 500 cells per square millimeter (range, 947 to 2833) in 11 of the 11 treated eyes (100%; 95% confidence interval [CI], 72 to 100), of which 10 had a CEC density exceeding 1000 cells per square millimeter. A corneal thickness of less than 630 μm (range, 489 to 640) was attained in 10 of the 11 treated eyes (91%; 95% CI, 59 to 100), and an improvement in best corrected visual acuity of two lines or more was recorded in 9 of the 11 treated eyes (82%; 95% CI, 48 to 98).

Conclusions: Injection of human CECs supplemented with a ROCK inhibitor was followed by an increase in CEC density after 24 weeks in 11 persons with bullous keratopathy. (Funded by the Japan Agency for Medical Research and Development and others; UMIN number, UMIN000012534 .)

https://pubmed.ncbi.nlm.nih.gov/29539291/