Generation of induced pluripotent stem cells from conjunctiva.
OBJECTIVE
The objective of this study was to determine whether cells from the conjunctiva could be reprogrammed into induced pluripotent stem (iPS) cells, providing an alternative source of stem cells.


METHODS
We employed a doxycycline-induced reprogrammable mouse strain to generate iPS cells from conjunctiva. The identity of the stem cells was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assays. Immunocytochemistry and teratoma assays are established means for scoring stem cell pluripotency. The reprogramming efficiencies of conjunctival cells and ear fibroblasts were compared.


RESULTS
We confirmed the identity of the stem cells and demonstrated expression of pluripotency markers (OCT4, SOX2, NANOG, and SSEA1), as tested by RT-PCR and immunofluorescence assays. In addition, derived iPS cells differentiated successfully into embryoid bodies, and showed teratoma formation when injected into immunodeficient mice. Reprogramming conjunctival tissue is as efficient as reprogramming ear fibroblasts. Conjunctiva-iPS exhibited classic features of embryonic stem (ES) cells with respect to morphology, expression of surface antigens, and pluripotency-associated transcription factors, capacity to differentiate in vitro, and the ability to form all three germ layers in vivo.


CONCLUSIONS
The present study demonstrated that conjunctival cells, which are readily obtained during the course of many routine conjunctival biopsies and ophthalmic procedures, can be another reliable source of iPS cells.
TianJin Medical University Eye Hospital, TianJin, 300384, China, yangjinchina324@gmail.com.
Jin
J
Yang
Yao
Y
Li
Deniz
D
Erol
Wen-Hsuan
WH
Wu
Yi-Ting
YT
Tsai
Xiao-Rong
XR
Li
Richard J
RJ
Davis
Stephen H
SH
Tsang
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D018920:Coculture Techniques; D003228:Conjunctiva / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D015415:Biomarkers; D018398:Homeodomain Proteins; D016256:Lewis X Antigen; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D004318:Doxycycline