Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting.
OBJECTIVE
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.
Ebrahimi
Marzeih
M
Department of Regenerative Medicine, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. mebrahimi@royaninstitute.org
Mohammadi
Parvaneh
P
Daryadel
Arezoo
A
Baharvand
Hossein
H
D000328:Adult; D000388:Air; D000650:Amnion / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D053558:Keratin-3 / Q000378:metabolism; D017770:Lifting; D008027:Light; D016850:Limbus Corneae / Q000166:cytology*; D012542:Scattering, Radiation; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*
D015415:Biomarkers; D018031:Connexin 43; D006360:Heat-Shock Proteins; D053558:Keratin-3
Immunity. 1998 Jun;8(6):657-65
9655479
Science. 1988 Sep 30;241(4874):1817-20
3175623
Stem Cells. 2006 Feb;24(2):368-75
16123387
Ann Med. 1999 Aug;31(4):261-71
10480757
Br J Ophthalmol. 2002 Apr;86(4):463-71
11914219
Exp Eye Res. 2005 Sep;81(3):247-64
16051216
Histol Histopathol. 2006 Sep;21(9):995-1027
16763950
Invest Ophthalmol Vis Sci. 2001 Nov;42(12):3031-40
11687552
Clin Exp Ophthalmol. 2001 Jun;29(3):138-42
11446453
Invest Ophthalmol Vis Sci. 2006 Jul;47(7):2823-7
16799020
Semin Cell Dev Biol. 2003 Oct;14(5):283-90
14986858
Handb Exp Pharmacol. 2006;(172):153-69
16610359
J Cataract Refract Surg. 2003 Jan;29(1):153-8
12551683
Exp Eye Res. 1991 Oct;53(4):545-8
1936190
Cell Tissue Res. 1991 Jun;264(3):497-506
1868521
Cell Biol Int. 2009 Sep;33(9):978-87
19559803
Exp Eye Res. 2008 Aug;87(2):96-105
18571161
J Neurosci. 1992 Aug;12(8):3004-9
1494943
Mol Vis. 2007 Sep 18;13:1711-21
17960109
Biochem Pharmacol. 2001 Jul 1;62(1):1-11
11377391
Dev Growth Differ. 2007 Apr;49(3):241-51
17394602
Differentiation. 1997 May;61(4):251-60
9203348
Br J Ophthalmol. 2001 Aug;85(8):996-1000
11466261
Regen Med. 2008 May;3(3):329-42
18462056
Invest Ophthalmol Vis Sci. 2003 Jul;44(7):3059-65
12824252
Redox Rep. 2009;14(4):147-53
19695121
Ophthalmic Res. 1999;31(3):203-9
10224503
Curr Eye Res. 1995 Dec;14(12):1125-33
8974842
Results Probl Cell Differ. 1991;17:58-70
1803423