A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Beau J. Fenner; Nur Zahirah B.M. Yusoff; Matthias Fuest; Lei Zhou; Francisco Bandeira; Howard Y. Cajucom-Uy; H. K. Tan; Jodhbir S. Mehta; Gary H.F. Yam

doi:10.1186/s40662-019-0155-0

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Beau J. Fenner, Nur Zahirah B.M. Yusoff, Matthias Fuest, Lei Zhou, Francisco Bandeira, Howard Y. Cajucom-Uy, H. K. Tan, Jodhbir S. Mehta, Gary H.F. Yam

Research output: Journal article publication › Journal article › Academic research › peer-review

7 Citations (Scopus)

Abstract

Background: Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods: Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results: AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion: AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Original language	English
Article number	30
Journal	Eye and Vision
Volume	6
Issue number	1
DOIs	https://doi.org/10.1186/s40662-019-0155-0
Publication status	Published - Dec 2019
Externally published	Yes

Keywords

Amnion extract
Corneal stromal keratocytes
Marker expression
Proteomics

ASJC Scopus subject areas

Ophthalmology
Health Professions (miscellaneous)

More information

10.1186/s40662-019-0155-0

Cite this

Fenner, B. J., Yusoff, N. Z. B. M., Fuest, M., Zhou, L., Bandeira, F., Cajucom-Uy, H. Y., Tan, H. K., Mehta, J. S., & Yam, G. H. F. (2019). A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy. Eye and Vision, 6(1), [30]. https://doi.org/10.1186/s40662-019-0155-0

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title = "A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy",

abstract = "Background: Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods: Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results: AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion: AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.",

keywords = "Amnion extract, Corneal stromal keratocytes, Marker expression, Proteomics",

author = "Fenner, {Beau J.} and Yusoff, {Nur Zahirah B.M.} and Matthias Fuest and Lei Zhou and Francisco Bandeira and Cajucom-Uy, {Howard Y.} and Tan, {H. K.} and Mehta, {Jodhbir S.} and Yam, {Gary H.F.}",

note = "Funding Information: We thank the Lion Eye Institute for Transplant and Research (Tampa, FL, US) for their assistance with procurement of research grade donor corneas. We also thank the Singapore Eye Bank, Singapore National Eye Centre, for the help in amnion collection (consent taking and tissue processing). We acknowledge SingHealth Advanced Bioimaging Core Facilities for confocal microscopy. Funding Information: Singapore National Eye Centre HREF 0618–2 and Singapore National Research Foundation under Clinician Scientist Award – Senior Investigator Category (JRNMRR163801), National Medical Research Council, Ministry of Health, Singapore. Publisher Copyright: {\textcopyright} 2019, The Author(s).",

year = "2019",

month = dec,

doi = "10.1186/s40662-019-0155-0",

language = "English",

volume = "6",

journal = "Eye and Vision",

issn = "2326-0246",

publisher = "Springer Verlag",

number = "1",

}

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy. / Fenner, Beau J.; Yusoff, Nur Zahirah B.M.; Fuest, Matthias et al.
In: Eye and Vision, Vol. 6, No. 1, 30, 12.2019.

Research output: Journal article publication › Journal article › Academic research › peer-review

TY - JOUR

T1 - A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

AU - Fenner, Beau J.

AU - Yusoff, Nur Zahirah B.M.

AU - Fuest, Matthias

AU - Zhou, Lei

AU - Bandeira, Francisco

AU - Cajucom-Uy, Howard Y.

AU - Tan, H. K.

AU - Mehta, Jodhbir S.

AU - Yam, Gary H.F.

N1 - Funding Information: We thank the Lion Eye Institute for Transplant and Research (Tampa, FL, US) for their assistance with procurement of research grade donor corneas. We also thank the Singapore Eye Bank, Singapore National Eye Centre, for the help in amnion collection (consent taking and tissue processing). We acknowledge SingHealth Advanced Bioimaging Core Facilities for confocal microscopy. Funding Information: Singapore National Eye Centre HREF 0618–2 and Singapore National Research Foundation under Clinician Scientist Award – Senior Investigator Category (JRNMRR163801), National Medical Research Council, Ministry of Health, Singapore. Publisher Copyright: © 2019, The Author(s).

PY - 2019/12

Y1 - 2019/12

N2 - Background: Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods: Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results: AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion: AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

AB - Background: Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods: Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results: AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion: AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

KW - Amnion extract

KW - Corneal stromal keratocytes

KW - Marker expression

KW - Proteomics

UR - http://www.scopus.com/inward/record.url?scp=85085276793&partnerID=8YFLogxK

U2 - 10.1186/s40662-019-0155-0

DO - 10.1186/s40662-019-0155-0

M3 - Journal article

AN - SCOPUS:85085276793

SN - 2326-0246

VL - 6

JO - Eye and Vision

JF - Eye and Vision

IS - 1

M1 - 30

ER -

A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Abstract

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