Value of human amniotic epithelial cells in tissue engineering for cornea

Abstract

Human amniotic epithelial cells (hAECs) are potentially one of the key players in tissue engineering due to their easy availability. The aim of the present study was to develop an optimal isolation and transportation technique, as well as to determine the immunophenotype and epithelial gene expression of hAECs. Amnion was mechanically peeled off from the chorion and digested with trypsin-ethylenediaminetetraacetic acid. The isolated hAECs were cultured in medium containing 10 ng/mL epidermal growth factor until P4. The epithelial gene expression, cell surface antigen and protein expression of hAECs were analyzed by quantitative polymerase chain reaction, flow cytometry and immunocytochemistry. hAECs were also cultured in adipogenic, osteogenic and neurogenic induction media. The best cell yield of hAECs was seen in the digestion of 15 pieces of amnion (2 × 2 cm) and isolated 30 min after digestion with trypsin. F12:Dulbecco's modified eagle medium was the best medium for short term storage at 4 °C. hAECs expressed CD9, CD44, CD73 and CD90, and negligibly expressed CD31, CD34, CD45 and CD117. After serial passage, CK3, CK19 and involucrin gene expressions were upregulated, while p63, CK1 and CK14 gene expressions were downregulated. Sustained gene expressions of integrin β1 and CK18 were observed. At initial culture, these cells might have stem-like properties. However, they differentiated after serial passage. Nonetheless, hAECs have epithelial stem cell characteristics and have the potential to differentiate into corneal epithelial cells. Further investigations are still needed to elucidate the mechanism of differentiation involved and to optimize the culture condition for long term in vitro culture.

Effects of epidermal growth factor on the proliferation and cell cycle regulation of cultured human amnion epithelial cells

“…This is similar with previous studies on other epithelial cells such as epidermal keratinocytes (11,12,16) and oropharyngeal keratinocytes (14). In some other studies, different concentrations of EGF were added to HAECs culture medium (2,3,7,8) to support the cells' proliferation. However, other growth supplements were also added to their culture medium such as fetal bovine serum, L-glutamine, PDGF, FGF, insulin and hydrocortisone (2,3,7,8).…”

“…In some other studies, different concentrations of EGF were added to HAECs culture medium (2,3,7,8) to support the cells' proliferation. However, other growth supplements were also added to their culture medium such as fetal bovine serum, L-glutamine, PDGF, FGF, insulin and hydrocortisone (2,3,7,8). Based on this study, it was found that 10 ng/ml EGF added to the culture medium could be the minimal concentration of EGF required to significantly increase the total number of cultured cells.…”

“…Additionally, they also showed that HAECs express the pluripotent and multipotent markers such as Oct-3/4, Sox2, Nanog, FGF-4, Rex-1, FZD-9, BST-1 and ABCG2 as well as neural stem cell markers such as nestin, vimentin and neuron specific enolase (NSE) (2e5). Their characterization and differentiation potentials into three germ layers (ectodermal-, mesodermal-and endodermal-lineages) have been thoroughly carried out (2,3). Although they have pluripotent characteristics, they do not form teratoma upon transplantation into the testes of the severe combined immunodeficiency (SCID) mice (2).…”

Cellular Response of Limbal Stem Cells on Poly (Hydroxybuthyrate-co-Hydroxyvalerate) Porous Scaffolds for Ocular Surface Bioengineering

“…Furthermore there are negative for KRT3=12, but were better for amniotic membrane and thermo responsive substrate than modified porous scaffold ( Figure 5). In recent years, many carriers have been used to culture limbal stem cells; however, the majority of previous studies have used amniotic membrane as a choice of carrier due to its low-cost procurement, easy availability, promote epithelialization, and reduced inflammation and scarring [30,31]. Nonetheless, due to its biologic origin, amniotic membrane carries inherent risks such as disease transmission and infection that cannot be totally avoided [32].…”

Limbal Epithelial Stem Cells in Corneal Regeneration

“…HAM is currently the most commonly used substrate for LESCs cultivation and transplantation. [38][39][40][41] Although the results are quite promising, amniotic membrane does have some shortcomings. One of the major issues is ensuring the biosafety of HAM in disease transmission, e.g., HIV, hepatitis B and C as well as from bacteria and fungus which will grow readily on HAM.…”