Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation

Journal of Translational Medicine

Marie-Jose Tassignon³  Zwi Berneman¹  Jean-Pierre Timmermans²  Carina Koppen³  Jos Rozema³  Inge Leysen³  Sorcha Ní Dhubhghaill³  Tine Possemiers³  Nadia Zakaria³ 

[1] Centre for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Edegem 2650, Belgium;Department of Veterinary Sciences, University of Antwerp, Edegem 2650, Belgium;Dept of Ophthalmology, Antwerp University Hospital, Edegem 2650, Belgium

关键词: Composite grafts;    CNT-20;    SHEM;    Corneal opacity;    Corneal neovascularization;    Ocular surface reconstruction;    Corneal reconstruction;    Limbal epithelial stem cells;    Somatic stem cells;    Progenitor cells;    Cell culture;    Cellular therapy;    Cell transplantation;    Tissue regeneration;    Tissue specific stem cells;    Amniotic membrane;    Clinical trial;    Limbal stem cell transplantation;   

Others  :  817789
DOI  :  10.1186/1479-5876-12-58

 received in 2013-11-12, accepted in 2014-02-19,  发布年份 2014

【 摘 要 】

Background

To determine if a standardized, non-xenogenic, reduced manipulation cultivation and surgical transplantation of limbal stem cell grafts is a safe and effective treatment option for patients with total and partial limbal stem cell deficiency.

Methods

In vitro cellular outgrowth and phenotype of the limbal epithelial cell and composite grafts were validated using a new protocol. Patients received either autologous (n = 15) or allogenic (n = 3) explants cultured using a standardized protocol free from xenogenic products. The resulting grafts were transplanted using a reduced manipulation surgical technique.

Results

The majority of cells (>50%) displayed a progenitor phenotype typified by positive immunofluorescence for ∆Np63, CK14 and ABCG2 and low immunofluorescence for CK3/12 and desmoglein 3 proteins. The surgical protocol was designed to minimize manipulation and the graft itself was secured without sutures. The transplant recipients were followed for a mean of 24 months. Twelve of the 18 transplant recipients were graded as anatomically successful (67%), based on the defined success parameters. There was a significant reduction in corneal neovascularization, which was accompanied by an improvement in pain though not photophobia or central corneal opacity post transplant. The transplantation protocol showed no measureable effect on visual acuity.

Conclusion

We conclude that this standardized culture system and surgical approach is safe and effective in reducing corneal neovascularization. The technique is free from animal contaminants and maintains a large proportion of progenitor cells. Although this technique did not improve visual function, restoring a functional epithelial cell layer and reducing corneal neovascularization provides an improved platform for a penetrating keratoplasty to ultimately improve visual function.

【 授权许可】

   
2014 Zakaria et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Puangsricharern V, Tseng SC: Cytologic evidence of corneal disease with limbal stem cell deficiency. Ophthalmology 1995, 102:1476-1485.
• [2]Rumelt S, Bersudsky V, Blum-Haruveni T, Rehany U: Systematic cyclosporing A in high failure risk, repeated corneal transplantation. Br J Ophthalmol 2002, 86:988-992.
• [3]Holland E: Epithelial transplantation for the management of severe ocular surface disease. Trans Am Ophthalmol Soc 1996, 94:677-743.
• [4]Shimmura S, Tsubota K: Surgical treatment of limbal stem cell deficiency: are we really transplanting stem cells? Am J Ophthalmol 2008, 146:154-155.
• [5]Daya S, Watson A, Sharpe JR, Giledi O, Rowe A, Martin R, James SE: Outcomes and DNA analysis of ex vivo expanded stem cell allograft for ocular surface reconstruction. Ophthalmology 2005, 112:470-477.
• [6]Kolli S, Ahmad S, Lako M, Figueiredo F: Successful clinical implementatio of corneal epithelial stem cell therapy for treatment of unilateral stem cell deficiency. Stem Cells 2010, 28:597-610.
• [7]Nakamura T, Inatomi T, Sotozono C, Ang LP, Koizumi N, Yokoi N, Kinoshita S: Transplantation of autologous serum-derived cultivated corneal epithelial equivalents for the treatment of severe ocular surface disease. Ophthalmology 2006, 113:1765-1772.
• [8]Pauklin M, Fuchsluger TA, Westekemper H, Steuhl KP, Meller D: Midterm results of cultivated autologous and allogenic limbal epithelial transplantation in limbal stem cell deficiency. Dev Ophthalmol 2010, 45:57-70.
• [9]Rama P, Matuska S, Paganoni G, Spinelli A, De LM, Pellegrini G: Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 2010, 363:147-155.
• [10]Sangwan A, Secker GA, Rajan MS, Melingonis G, Dart JK, Tuft SJ, Daniels JT: Ex vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology 2008, 115:1989-1997.
• [11]Sangwan A, Matalia HP, Vemuganti GK, Fatima A, Ifthekar G, Singh S, Nutheti R, Rao GN: Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian J Ophthalmology 2006, 54:29-34.
• [12]Koizumi N, Inatomi T, Suzuki T, Sotozono C, Kinoshita S: Cultivated corneal epithelial stem cell transplantation in ocular surface disorders. Ophthalmology 2001, 108:1569-1574.
• [13]Shimazaki J, Higa K, Morito F, Dogru M, Kawakita T, Satake Y, Shimmura Y, Tsubota K: Factors influencing outcomes in cultivated limbal epithelial transplantation for chronic cicatricial ocular surface disorders. Am J Ophthalmol 2007, 143:945-953.
• [14]Sangwan V: Basu Sm Vemuganti GK et al. Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study. Br J Ophthalmol 2011, 95:1525-1529.
• [15]Zakaria N, Koppen C, Van Marck V, Berneman Z, Hopkinson A, Tassignon MJ: Standardized limbal epithelial stem cell graft generation and transplantation. Tissue Eng Part C: Methods 2010, 16:921-927.
• [16]Donisi P, Rama P, Fasolo A, Ponzin D: Analysis of limbal stem cell deficiency be corneal impression cytology. Cornea 2003, 22:533-538.
• [17]Schulze-Bonsel KF,F, Burau H, Hansen L, Bach M: Visual acuities “hand motion” and “counting fingers” can be quantified with the Freiburg Visual Acuity Test. Invest Ophthalmol Vis Sci 2006, 47:1236-1240.
• [18]Flaherty S: Pain measurement tools for clinical practice and research. AANA J 1996, 64:133-140.
• [19]Hopkinson A, McIntosh RS, Tighe PJ, James DK, Dua HS: Amniotic membrane for ocular reconstruction: donor variation and the effect of handling on TGF-beta content. Invest Ophthalmol Vis Sci 2006, 47:4316-4322.
• [20]Hopkinson A, Shanmuganathan VA, Gray T, Yeung AM, Lowe J, James DK, Dua HS: Optimization of amniotic membrane denuding for tissue engineering. Tissue Eng Part C: Methods 2008, 14:371-381.
• [21]Zakaria N, Van GS, Wouters K, Rozema J, Koppen C, Lion E, Cools N, Berneman Z, Tassignon MJ: Human tears reveal insights into corneal neovascularization. PLoS One 2012, 7:e36451.
• [22]Schallmoser K, Rohde E, Reinisch A, Bartmann C, Thaler D, Drexler C, Obenauf AC, Lanzer G, Linkesch W, Strunk D: Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C: Methods 2008, 14:185-196.
• [23]Report. U.S. Department of Health and Human Services: Food and Drug Administration (FDA) centre for biologics evaluation and research (CBER). In Information for recommendations for physicians involved in the co-culture of human embryos with nonhuman animal cells. Rockville MD: US (FDA) Report; 2000.
• [24]Martin M, Muotri A, Gage F, Varki A: Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005, 11:228-232.
• [25]Lekhanont K, Choubtum L, Chuch RS, Sangiampornpanit T, Chuchpaiwong V, Vongthongsri A: A serum and feeder-free technique of culturing human corneal epithelial stem cells on amniotic membrane. Mol Vis 2009, 15:1294-1302.
• [26]Gstraunthaler G: Alternatives to the use of fetal bovine serum. ALTEX 2003, 20:275-281.
• [27]Shortt A, Secker CA, Notara MD, Limb GA, Khaw PT, Tuft SJ, Daniels JT: Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007, 52:371-379.
• [28]Panda A, Kumar S, Kumar A, Bansal R, Bhartiya S: Fibrin glue in ophthalmology. Indian J Ophthalmology 2009, 57:371-379.
• [29]Nassiri N, Pandya HK, Djalillian AR: Limbal allograft transplantation using fibrin glue. Arch Ophthalmol 2011, 129:218-222.
• [30]Takaoka M, Nakamura T, Sugai H, Bentley AJ, Nakajima N, Yokoi N, Fullwood : Novel sutureless keratoplasty with chemically defined bioadhesive. Invest Ophthalmol Vis Sci 2009, 50:2679-2685.
• [31]Shortt AJTS, Daniels JT: Ex vivo cultured limbal epithelial transplantation: a clinical perspective. Ocul Surf 2010, 8:80-90.
• [32]Ni Dhubhghaill S, Rozema JJ, Jongenelen S, Ruiz Hidalgo I, Zakaria N, Tassignon MJ: Normative values for corneal densitometry analysis by scheimpflug optical assessment. Invest Ophthalmol Vis Sci 2014, 55:162-168.
• [33]Hillenaar T, Cals RH, Eilers PH, Wubbels RJ, van Cleynenbreugel H, Remeijer L: Normative database for corneal backscatter analysis by in vivo confocal microscopy. Invest Ophthalmol Vis Sci 2011, 52:7274-7281.
• [34]Wang J, Simpson TL, Fonn D: Objective measurements of corneal light-backscatter during corneal swelling by optical coherence tomography. Invest Ophthalmol Vis Sci 2004, 45:3493-3498.