Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting

Abstract

Objectives: The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure.

Methods: Limbal biopsies taken from cadaveric normal human limbus were cultivated as explants on human amniotic membrane (HAM) and plastic dish (PD). Corneal differentiation was induced by air lifting for 16 days. The expression of putative LSC markers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers (keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90, and HSC70 were tested by RT-PCR, immunofluorescence, and flow cytometry pre- and post-air exposure. Fresh limbal and corneal tissues were used as control groups.

Results: Air lifting induced corneal differentiation with a decrease in the number of P63(+) cells and an increase in the number of K3(+)/CX43(+) cells, which characterized transient amplifying cells (TACs). Moreover, denuded HAM provided a superior niche for LSC proliferation and phenotype maintenance in vitro. Additionally, we have evidence that expressions of HSC70 as well as HSP72 were enhanced through corneal differentiation and HSP90 post-air lifting in vitro and in vivo. HSP60, however, was not detected in either LSC or corneal cells, in vivo and in vitro.

Conclusions: These results suggest that corneal differentiation following air exposure may regulate HSP72 and HSC70 expression. In addition, HSP72 and HSP90 may protect LSC and corneal cells against oxidative stress.

Figure 1

Human limbal explants expanded on human amniotic membrane (HAM) and plastic dish (PD) for 14 days, followed by air lifting for 16 days. A: Phase-contrast microscopy of the cells shows that cells on HAM are small and compact compared with those in PD pre air lifting; however, the morphology of the cells in the PD group changes to squamous epithelial cells post air lifting. Scale bar: 100 µm. B: Assessment of cell heterogeneity in R1: 10–200 or limbal stem cell (LSC), R2: 200–400 or Transient amplifying cells (TAC), and R3: >400 or terminal differentiated cells (TDC) by light scatter showed that cells in PD are more differentiated than those in AM. C: The comparison of cells in PD and AM groups before and after air lifting shows cells differentiated to large cells with high granularity. Results were presented as percentage of 3 different experiments and expressed as mean±SD. The astereisk indicates a p<0.05 and the double asterisk indicates a p<0.01.

Figure 2

Evaluation of limbal stem cell and corneal differentiation markers pre and post air lifting in cultured limbal cells on AM and PD groups. A: Representative fluorescent staining profile shows that P63, a putative stemness marker, expressed in nucleus and K3, a differentiation marker, in cytoplasm and Cx43 in cell membrane and near the nucleus in cultured cells on amniotic membrane (AM) and plastic dish (PD) before and after air lifting. The nuclei were stained by Dappi (Blue). Scale bar: 100 µm. B: Comparative data for PD and AM group for P63, K3, and CX43, before and after air lifting by flowcytometry analysis. C: Immuno staining of cornea and limbal tissues for P63 and K3 Marker. Scale bar: 100 µm. D: RT–PCR shows the gene expression of P63,K3, ABCG2 and GAPDH as internal control before and after air lifting in AM, PD as well as limbal and corneal cells. Results were presented as percentage of 3 to 4 different experiments and expressed as mean±SD. The asterisk indicates a p<0.05 and the double asterisk indicates a p<0.01.

Figure 3

Evaluation of HSPs pre and post air lifting in AM and PD groups. A: Representative fluorescent staining indicates HSC70, HSP72, and HSP90 are expressed in cytoplasm. HSP72 also detected in few nuclei of cells cultured on amniotic membrane before and after air lifting. The nuclei were stained by DAPI (Blue). Scale bar: 100 µm. B: Representative histograms show the percentage of cells positive for HSC70, HSP72, and HSP90 on plastic dish. Black line indicates isotype control and red points out HSPs. C: Comparative data for PD and AM groups for HSPs before and after air lifting. D: RT–PCR shows the gene expression of HSC70, HSP72, and HSP90 before and after air lifting in AM and PD. GAPDH was used as internal control. Results were presented as percentage of 3 to 4 different experiments and expressed as mean±SD. The asterisk indicates a p<0.05 and the double asterisk indicates a p<0.01.