Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells

Muhammad Ali, Shahid Khan, Shivakumar Vasanth, Mariya R. Ahmed, Ruiqiang Chen, Chan-Hyun Na, Jason J. Thomson, Caihong Qiu, John D Gottsch, Sheikh Amer Riazuddin

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2437-2444
Number of pages8
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number6
DOIs
StatePublished - May 1 2018

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Induced Pluripotent Stem Cells
Proteome
Blood Cells
Endothelial Cells
Corneal Endothelium
Phase-Contrast Microscopy
Proteins
Neural Crest
Cryopreservation

Keywords

  • Based proteome sequencing
  • Corneal endothelial cells
  • Induced pluripotent stem cells
  • Mass-spectrometry

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells. / Ali, Muhammad; Khan, Shahid; Vasanth, Shivakumar; Ahmed, Mariya R.; Chen, Ruiqiang; Na, Chan-Hyun; Thomson, Jason J.; Qiu, Caihong; Gottsch, John D; Riazuddin, Sheikh Amer.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 6, 01.05.2018, p. 2437-2444.

Research output: Contribution to journalArticle

Ali, Muhammad ; Khan, Shahid ; Vasanth, Shivakumar ; Ahmed, Mariya R. ; Chen, Ruiqiang ; Na, Chan-Hyun ; Thomson, Jason J. ; Qiu, Caihong ; Gottsch, John D ; Riazuddin, Sheikh Amer. / Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 6. pp. 2437-2444.
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T1 - Generation and proteome profiling of PBMC-originated, iPSC-derived corneal endothelial cells

AU - Ali, Muhammad

AU - Khan, Shahid

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, Sheikh Amer

PY - 2018/5/1

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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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