Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts

Jennifer L Badger, Meg L Byrne, Farlan S Veraitch, Chris Mason, Ivan B Wall, Maeve A Caldwell

Research output: Contribution to journalArticle

Abstract

AIM: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate.

MATERIALS & METHODS: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A.

RESULTS: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers.

CONCLUSION: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

Original languageEnglish
Pages (from-to)675-683
Number of pages9
JournalRegenerative medicine
Volume7
Issue number5
DOIs
Publication statusPublished - Sep 2012

Fingerprint

Pluripotent Stem Cells
Fibroblasts
Stem cells
Cell culture
Cell Line
Coculture Techniques
Oxygen
Feeder Cells
Germ Layers
Blastocyst
Transcription factors
Embryonic Stem Cells
Hypoxia
Adenosinetriphosphate
Research
Cultured Cells
Respiration
Transcription Factors
Stem Cells
Cell Culture Techniques

Keywords

  • Animals
  • Biomarkers/metabolism
  • Cell Culture Techniques/methods
  • Cell Extracts
  • Cell Hypoxia
  • Cell Line
  • Cell Proliferation
  • Cell Survival
  • Embryo, Mammalian/cytology
  • Fibroblasts/cytology
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
  • Mice
  • Pluripotent Stem Cells/cytology
  • Protein Stability

Cite this

Badger, Jennifer L ; Byrne, Meg L ; Veraitch, Farlan S ; Mason, Chris ; Wall, Ivan B ; Caldwell, Maeve A. / Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts. In: Regenerative medicine. 2012 ; Vol. 7, No. 5. pp. 675-683.
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Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts. / Badger, Jennifer L; Byrne, Meg L; Veraitch, Farlan S; Mason, Chris; Wall, Ivan B; Caldwell, Maeve A.

In: Regenerative medicine, Vol. 7, No. 5, 09.2012, p. 675-683.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts

AU - Badger, Jennifer L

AU - Byrne, Meg L

AU - Veraitch, Farlan S

AU - Mason, Chris

AU - Wall, Ivan B

AU - Caldwell, Maeve A

PY - 2012/9

Y1 - 2012/9

N2 - AIM: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate.MATERIALS & METHODS: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A.RESULTS: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers.CONCLUSION: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

AB - AIM: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate.MATERIALS & METHODS: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A.RESULTS: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers.CONCLUSION: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

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KW - Cell Culture Techniques/methods

KW - Cell Extracts

KW - Cell Hypoxia

KW - Cell Line

KW - Cell Proliferation

KW - Cell Survival

KW - Embryo, Mammalian/cytology

KW - Fibroblasts/cytology

KW - Humans

KW - Hypoxia-Inducible Factor 1, alpha Subunit/metabolism

KW - Mice

KW - Pluripotent Stem Cells/cytology

KW - Protein Stability

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DO - 10.2217/rme.12.55

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JF - Regenerative medicine

SN - 1746-0751

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