Corneal Epithelial Stem Cells: Methods for Ex Vivo Expansion

  • Gustavo S. Figueiredo
  • Hardeep Singh Mudhar
  • Majlinda Lako
  • Francisco C. FigueiredoEmail author
Part of the Essentials in Ophthalmology book series (ESSENTIALS)


The corneal epithelium in humans is the only layer in the cornea that is able to regenerate in vivo, and corneal integrity and function are dependent on this. The ability of the epithelium to regenerate relies on limbal stem cells (LSCs). The LSC niche has been demonstrated to be found within the palisades of Vogt at the corneoscleral limbus. Total limbal stem cell deficiency (LSCD) results in conjunctivalisation of the corneal surface and subsequent permanent visual impairment, together with recurrent or persistent epithelial erosions and ulcerations that lead to stromal opacity and chronic pain and discomfort. In addition to conservative management with lubricating eye drops, serum eye drops, topical steroid and antibiotics, the definitive treatment of total LSCD involves replacing the LSC population before patients can undergo corneal transplantation for visual rehabilitation. This chapter describes the Newcastle methods of autologous limbal stem cell transplantation from the harvesting and culture of autologous LSCs to transplantation of the medicinal product, using a cell suspension method or ex vivo expansion method, describes the Good Manufacturing Practice (GMP) processes for the production of cells for therapeutic use and looks to the future use of a European-licensed LSC therapeutic product (Holoclar®, Chiesi Farmaceutici S.p.A., Parma, Italy) for the treatment of unilateral severe or total LSCD due to ocular surface burns. This chapter also describes the methods for the assessment of the LSC culture, including histological methods, and the potential use of decellularised, γ-irradiated human amniotic membrane as a suitable substrate for LSC culture.


Limbal stem cells Limbal stem cell deficiency Stem cell culture Stem cell transplantation Cell therapy Good Manufacturing Practice Human amniotic membrane 


Compliance with Ethical Requirements

Gustavo S. Figueiredo, Hardeep S. Mudhar, Majlinda Lako and Francisco C. Figueiredo declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study. No animal studies were performed by the authors for this chapter.


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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Gustavo S. Figueiredo
    • 1
  • Hardeep Singh Mudhar
    • 2
  • Majlinda Lako
    • 1
  • Francisco C. Figueiredo
    • 1
    • 3
    Email author
  1. 1.Newcastle University, Institute of Genetic Medicine, International Centre for LifeNewcastle upon TyneUK
  2. 2.Royal Hallamshire Hospital, Department of HistopathologySheffieldUK
  3. 3.Royal Victoria InfirmaryNewcastle upon TyneUK

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