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Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells

Ellen Scheerlinck, Katleen Van Steendam, Mado Vandewoestyne UGent, Trees Lepez UGent, Veerle Gobin, Paulien Meert, Liesbeth Vossaert UGent, Filip Van Nieuwerburgh UGent, Ann Van Soom UGent, Luc Peelman UGent, et al. (2014) ANALYTICAL BIOCHEMISTRY. 461. p.60-66
abstract
The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs).
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
PLURIPOTENCY, OCT4, SELF-RENEWAL, MAMMALIAN-CELLS, MEF CM, CULTURE, OCT4, Flow cytometry, Fluorescence microscopy, OCT4-eGFP knock-in hESC
journal title
ANALYTICAL BIOCHEMISTRY
Anal. Biochem.
volume
461
pages
60 - 66
Web of Science type
Article
Web of Science id
000340077600010
JCR category
CHEMISTRY, ANALYTICAL
JCR impact factor
2.219 (2014)
JCR rank
32/74 (2014)
JCR quartile
2 (2014)
ISSN
0003-2697
DOI
10.1016/j.ab.2014.05.026
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
5719475
handle
http://hdl.handle.net/1854/LU-5719475
date created
2014-10-09 13:46:12
date last changed
2016-12-19 15:39:18
@article{5719475,
  abstract     = {The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs).},
  author       = {Scheerlinck, Ellen and Van Steendam, Katleen and Vandewoestyne, Mado and Lepez, Trees and Gobin, Veerle and Meert, Paulien and Vossaert, Liesbeth and Van Nieuwerburgh, Filip and Van Soom, Ann and Peelman, Luc and Heindryckx, Bj{\"o}rn and De Sutter, Petra and Dhaenens, Maarten and Deforce, Dieter},
  issn         = {0003-2697},
  journal      = {ANALYTICAL BIOCHEMISTRY},
  keyword      = {PLURIPOTENCY,OCT4,SELF-RENEWAL,MAMMALIAN-CELLS,MEF CM,CULTURE,OCT4,Flow cytometry,Fluorescence microscopy,OCT4-eGFP knock-in hESC},
  language     = {eng},
  pages        = {60--66},
  title        = {Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells},
  url          = {http://dx.doi.org/10.1016/j.ab.2014.05.026},
  volume       = {461},
  year         = {2014},
}

Chicago
Scheerlinck, Ellen, Katleen Van Steendam, Mado Vandewoestyne, Trees Lepez, Veerle Gobin, Paulien Meert, Liesbeth Vossaert, et al. 2014. “Detailed Method Description for Noninvasive Monitoring of Differentiation Status of Human Embryonic Stem Cells.” Analytical Biochemistry 461: 60–66.
APA
Scheerlinck, Ellen, Van Steendam, K., Vandewoestyne, M., Lepez, T., Gobin, V., Meert, P., Vossaert, L., et al. (2014). Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells. ANALYTICAL BIOCHEMISTRY, 461, 60–66.
Vancouver
1.
Scheerlinck E, Van Steendam K, Vandewoestyne M, Lepez T, Gobin V, Meert P, et al. Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells. ANALYTICAL BIOCHEMISTRY. 2014;461:60–6.
MLA
Scheerlinck, Ellen, Katleen Van Steendam, Mado Vandewoestyne, et al. “Detailed Method Description for Noninvasive Monitoring of Differentiation Status of Human Embryonic Stem Cells.” ANALYTICAL BIOCHEMISTRY 461 (2014): 60–66. Print.