TY - JOUR
T1 - Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells
AU - Ali, Muhammad
AU - Khan, Shahid Y.
AU - Vasanth, Shivakumar
AU - Ahmed, Mariya R.
AU - Chen, Ruiqiang
AU - Na, Chan Hyun
AU - Thomson, Jason J.
AU - Qiu, Caihong
AU - Gottsch, John D.
AU - Riazuddin, S. Amer
N1 - Funding Information:
Supported in part by National Eye Institute Grant R01EY028538 and the Kwok Research Fund. Disclosure: M. Ali, None; S.Y. Khan, None; S. Vasanth, None; M.R. Ahmed, None; R. Chen, None; C.H. Na, None; J.J.
Publisher Copyright:
© 2018 The Authors.
PY - 2018/5
Y1 - 2018/5
N2 - PURPOSE. Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. METHODS. We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry– based proteome sequencing. RESULTS. The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry–based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. CONCLUSIONS. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.
AB - PURPOSE. Corneal endothelial cells (CECs) are critical in maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived CECs. METHODS. We isolated PBMCs and programmed the mononuclear cells to generate iPSCs, which were differentiated to CECs through the neural crest cells (NCCs). The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent and corneal endothelium (CE)-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSC-derived CECs and human corneal endothelium (hCE) was examined by mass spectrometry– based proteome sequencing. RESULTS. The PBMC-originated, iPSC-derived CECs were tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CE-associated markers expressed at significantly higher levels in iPSC-derived CECs at days 13, 20, and 30 compared with their respective levels in iPSCs. It is of importance that only residual expression levels of pluripotency markers were detected in iPSC-derived CECs. Cryopreservation of iPSC-derived CECs did not affect the tight adherence of CECs and their hexagonal-like shape while expressing high levels of CE-associated markers. Mass spectrometry–based proteome sequencing identified 10,575 proteins in the iPSC-derived CEC proteome. In parallel, we completed proteome profiling of the hCE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSC-derived CECs, suggesting that 90.82% of the hCE proteome overlaps with the iPSC-derived CEC proteome. CONCLUSIONS. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the hCE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSC-derived CECs.
KW - Based proteome sequencing
KW - Corneal endothelial cells
KW - Induced pluripotent stem cells
KW - Mass-spectrometry
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U2 - 10.1167/iovs.17-22927
DO - 10.1167/iovs.17-22927
M3 - Article
C2 - 29847650
AN - SCOPUS:85047323464
SN - 0146-0404
VL - 59
SP - 2437
EP - 2444
JO - Investigative Ophthalmology and Visual Science
JF - Investigative Ophthalmology and Visual Science
IS - 6
ER -