Abstract
Transplantation of cultivated limbal epithelial transplantation has been proven to restore the corneal surface in limbal stem cell deficiency (LSCD). Here we comparatively investigated the optimized conditions and the efficiency of limbal epithelial sheet growth in three media conditions as well as with substrate free (transwell), human amniotic membrane (HAM) sutured onto transwell inserts (HAMTW), and HAM slide scaffold (HAMS). Outcomes evaluated were outgrowth sheet size from limbal explants, expression of stem/progenitor cell markers p63α, ABCG2 and CK15, and colony formation efficiency (CFE). Additionally, limbal epithelial sheets on HAMS were transplanted into corneas of LSCD rabbit models. Limbal epithelial sheets with 5% human AB serum showed the greatest increase in ABCG2 efflux activity (JC1low), p63α expression, and CFE compared in both conditions without HAM and with HAM, respectively. The outgrowth sheet size, cell yield, and Ki67 expression were increased in limbal epithelial sheets on HAMS compared to transwell and HAMTW. ABCG2 efflux activity, p63α and CK15 expressions, and CFE were also increased in limbal epithelial sheets on HAMS as well. In corneas of transplanted rabbit LSCD models, p63α expressions were noted in the basal layers and CK12 expressions were observed in superficial layers. Cultivation of limbal epithelial sheet on HAMS with xeno-free medium enhances the growth and stemness of limbal epithelial sheets.
Highlights
Limbal stem cells (LSCs) are essential to maintain cornea epithelium and endowed with a capacity for self-renewal and extended proliferative potential[1,2]
JC1low exclusion percentages of all three conditions on human amniotic membranes (HAM) were maintained above 20.3% and those in condition III induced a more than 8% increase in JC1low compared to two other conditions
To expand the phenotypic characterization of the effect of HAM slide scaffold (HAMS), we investigated the expression of limbal stem/progenitor cell markers, ATP-binding cassette sub-family G member 2 (ABCG2), p63α, cytokeratin 15 (CK15) and the corneal epithelial differentiation marker cytokeratin 12 (CK12)
Summary
Limbal stem cells (LSCs) are essential to maintain cornea epithelium and endowed with a capacity for self-renewal and extended proliferative potential[1,2]. Dysfunction or loss of these cells by chemical or thermal injury, radiation, repeated surgical interventions, and immunological disorders, results in limbal stem cell deficiency (LSCD) This is characterized by loss of corneal clarity, conjunctivalization, and visual loss[3]. Investigation of optimal culture conditions for increased percentile of stem/progenitor cells with regenerative capacity within limbal epithelial sheets is critically important to treat LSCD. Toward this goal, we comparatively investigated the proliferation potential and stemness of limbal epithelial sheets on substrate free and amniotic membrane scaffolds with xenobiotic free medium and demonstrated limbal epithelial sheet on HAM slide scaffold (HAMS) enhances the growth and stemness of limbal epithelial sheets
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