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

Ellen Scheerlinck (UGent) , Katleen Van Steendam (UGent) , Mado Vandewoestyne (UGent) , Trees Lepez (UGent) , Veerle Gobin (UGent) , Paulien Meert (UGent) , Liesbeth Vossaert (UGent) , Filip Van Nieuwerburgh (UGent) , Ann Van Soom (UGent) , Luc Peelman (UGent) , et al.
(2014) ANALYTICAL BIOCHEMISTRY. 461. p.60-66
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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).
Keywords
PLURIPOTENCY, OCT4, SELF-RENEWAL, MAMMALIAN-CELLS, MEF CM, CULTURE, OCT4, Flow cytometry, Fluorescence microscopy, OCT4-eGFP knock-in hESC

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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.
@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},
}

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