Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium.
OBJECTIVE
To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro.


METHODS
Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet's membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT-PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na(+)-K(+)-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na(+)-K(+)-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT-PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry.


RESULTS
In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3-4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na(+)-K(+)-ATPase, VDAC3, SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, and percentage of cells entering the S and G(2) phases were higher in the 25%ESC-CM group than in the CEM group. The number of apoptotic cells and p21 protein expression both decreased in the 25%ESC-CM group.


CONCLUSIONS
Use of 25%ESC-CM significantly increased the number of proliferating cells. These effects may be achieved through inhibition of p21 expression and apoptosis. These results suggested that 25%ESC-CM may be a new tool for cultivating HCECs for transplantation.
Lu
Xiaoyan
X
State Key laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, PR China.
Chen
Dong
D
Liu
Zhiping
Z
Li
Chaoyang
C
Liu
Ying
Y
Zhou
Jin
J
Wan
Pengxia
P
Mou
Yong-gao
YG
Wang
Zhichong
Z
D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D010750:Phosphoproteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D055815:Young Adult; D062826:Zonula Occludens-1 Protein
D017077:Culture Media, Conditioned; D050759:Cyclin-Dependent Kinase Inhibitor p21; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase
Exp Eye Res. 1999 May;68(5):531-9
10328966
Exp Eye Res. 1988 Sep;47(3):403-14
3053224
J Refract Surg. 2005 Jul-Aug;21(4):339-45
16128330
Cornea. 2001 Apr;20(3):239-43
11322409
Br J Ophthalmol. 1996 Mar;80(3):256-62
8703865
Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5545-9
7777546
Invest Ophthalmol Vis Sci. 2000 Sep;41(10):2930-5
10967047
Mol Vis. 2009;15:747-61
19381346
Invest Ophthalmol Vis Sci. 2006 Oct;47(10):4330-40
17003423
Tissue Eng Part C Methods. 2010 Aug;16(4):793-802
19842914
Am J Physiol Heart Circ Physiol. 2008 Aug;295(2):H907-13
18552162
Invest Ophthalmol Vis Sci. 1997 May;38(6):1119-29
9152231
Invest Ophthalmol Vis Sci. 1994 Jan;35(1):150-61
8300342
Exp Eye Res. 2004 Aug;79(2):231-7
15325570
Stem Cells. 2006 Apr;24(4):850-6
16339641
Proc Natl Acad Sci U S A. 2010 Mar 30;107(13):5845-50
20231440
Neuroreport. 2006 Jul 17;17(10):981-6
16791088
Oncogene. 1997 Feb 27;14(8):929-35
9050992
J Clin Invest. 1993 Apr;91(4):1713-20
8386193
Prog Retin Eye Res. 2003 May;22(3):359-89
12852491
Cornea. 2002 May;21(4):415-8
11973393
Cornea. 1993 May;12(3):249-54
8500339
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1387-96
16565372
Cell Cycle. 2004 Mar;3(3):380-3
14739780
Invest Ophthalmol Vis Sci. 1997 Sep;38(10):1922-8
9331255
Brain Res. 2000 Jun 30;869(1-2):130-6
10865067
Cornea. 2001 Jan;20(1):59-63
11189006
Invest Ophthalmol Vis Sci. 1993 Jul;34(8):2544-61
8392040
Am J Anat. 1990 Mar;187(3):254-60
2321558
Genes Dev. 1996 Aug 1;10(15):1945-52
8756351
Invest Ophthalmol Vis Sci. 2004 Sep;45(9):2992-7
15326112
Invest Ophthalmol Vis Sci. 2000 Mar;41(3):660-7
10711678
Invest Ophthalmol Vis Sci. 2004 Jul;45(7):2163-7
15223790
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2572-83
9856767
Cornea. 2001 Oct;20(7):731-7
11588426
Acta Ophthalmol Scand. 2000 Apr;78(2):130-6
10794243
Invest Ophthalmol Vis Sci. 2004 Jun;45(6):1743-51
15161835
Invest Ophthalmol Vis Sci. 1995 Jan;36(1):32-40
7822156
Ophthalmic Res. 1996;28(6):361-4
9032795
Invest Ophthalmol Vis Sci. 1993 May;34(6):2112-23
8387979
Nature. 1988 Dec 15;336(6200):688-90
3143917
Mol Vis. 2008 Sep 15;14:1705-12
18806879
Exp Eye Res. 2007 Mar;84(3):599-609
17223104
Invest Ophthalmol Vis Sci. 2002 Jul;43(7):2152-9
12091410
Genes Dev. 1994 Jan;8(1):9-22
8288131
Cell. 1993 Nov 19;75(4):805-16
8242751
Stem Cells. 2005 Oct;23(9):1324-32
16210409
Cell. 2008 Oct 31;135(3):449-61
18984157
Invest Ophthalmol Vis Sci. 1991 Sep;32(10):2747-56
1716619
Invest Ophthalmol Vis Sci. 2008 Apr;49(4):1392-7
18385055
Nature. 1993 Dec 16;366(6456):701-4
8259214
Mol Cancer Ther. 2003 Aug;2(8):773-82
12939467