Tese
Desenvolvimento e caracterização de córneas humanas descelularizadas e recelularização com células-tronco visando regeneração do epitélio corneano anterior
Fecha
2017-07-25Autor
Thaís Maria da Mata Martins
Institución
Resumen
Numerous diseases can affect corneal structure, leading to visual impairment and even
blindness. Currently, the only treatment for most corneal illnesses is transplantation of corneal
allografts. However, graft rejection and the lack of donors are the two major disadvantages
associated to this procedure. Generation of artificial corneas based on decellularization
methods is a promising alternative to overcome these problems. Therefore, the present study
aimed to develop and characterize decellularized human corneas, recellularize these
decellularized corneas (CDs) with human stem cells and promote the differentiation of these
cells into corneal epithelial-like cells (CECs) in culture medium supplemented with allogeneic
human serum (SH) replacing fetal bovine serum. Two decellularization protocols were tested:
1- sodium chloride (NaCl) treatment, 2 - NaCl plus nucleases treatment. The success of each
method on the removal of cells from the cornea and the integrity of the extracellular matrix
were investigated by histology, electron microscopy, DNA quantification,
immunofluorescence and nuclear staining with Hoechst. The CDs were recellularized with
human orbital fat-derived stem cells (OFSCs), human embryonic stem cells (hESCs), or
human induced pluripotent stem cells (hiPSCs) and the biocompatibility of the CDs was
analyzed by calcein-AM staining. The ability of the OFSCs, hESCs and hiPSCs to
differentiate into CECs when cultured on CDs or on laminin in culture medium supplemented
with SH was evaluated. The results showed that corneas processed using NaCl resulted in
incomplete removal of cellular material. In contrast, corneas decellularized with NaCl plus
nucleases method resulted in total removal of the cellular components. This treatment also
kept the epithelial basement membrane and stroma completely intact. Calcein-AM staining
demonstrated the viability, adhesion and a normal morphology of the OFSCs, hESCs and
hiPSCs seeded on CDs. OFSCs did not differentiate towards the CECs lineage, while hESCs
and hiPSCs differentiated into terminally differentiated CECs when seeded on CDs. These
results showed that NaCl plus nucleases treatment of human corneas generates an acellular
and biocompatible matrix with adequate histologic properties which have potential
applications in corneal tissue engineering.