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Stem cells and development2014; 23(13); 1515-1523; doi: 10.1089/scd.2013.0461

Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts.

Whitworth, DJ
·
Ovchinnikov, DA
·
Sun, J
·
Fortuna, PR
·
Wolvetang, EJ

Abstract: In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse.

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Publication Date: 2014-04-01 PubMed ID: 24555755PubMed Central: PMC4066230DOI: 10.1089/scd.2013.0461Google Scholar: Lookup

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This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The researchers in this study have managed to create pluripotent stem cells from adult horse skin cells that are dependent only on a factor called leukemia inhibitory factor. This contrasts with previous attempts where two factors were needed. These created stem cells are normal, stable, and can develop into a range of tissue types which might further our understanding of pluripotency in horses.

Creation of equine induced pluripotent stem cells (equiPSCs)

The scientists reprogrammed adult female horse skin cells, termed ‘dermal fibroblasts’, into equine pluripotent stem cells.
These equiPSCs need only the leukemia inhibitory factor (LIF) to sustain them, a significant shift from prior derived equiPSCs that required both LIF and basic fibroblast growth factor (bFGF).

Characterization of equiPSCs

Generated equiPSCs were found to possess a normal karyotype, indicating they have a normal number and arrangement of chromosomes.
These cells have been stable and maintained for over 60 cycles of replication.
They exhibit alkaline phosphatase activity, a common characteristic of stem cells.
They express certain proteins (eqNANOG, eqOCT4, and eqTERT mRNA), evidence of their pluripotent nature.
Immunocytochemistry showed that they produce additional proteins (NANOG, REX1, SSEA4, TRA1-60, and TRA1-81) which are related to pluripotency.

Role of basic fibroblast growth factor (bFGF) and X chromosome Reactivation

Despite the equiPSCs being maintained just with LIF, the scientists found that bFGF aids their proliferation through a specific biochemical pathway, the PI3K/AKT pathway.
The researchers also noted a lack of expression of eqXIST and absence of a certain mark, H3K27me3, suggesting reactivation of the inactive X chromosome during reprogramming. Nothing that cells have two active X chromosomes indicates a more naive state of pluripotency.

Potential of equiPSCs

The cells are capable of forming embryoid bodies and in-vitro teratomas, which contain derivatives of all three germ layers, demonstrating their pluripotent nature.
This illustrates potential to develop into a wide variety of tissue types.
The distinct LIF-dependent equiPSCs offer a new resource for understanding pluripotency in the horse.

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APA

Whitworth DJ, Ovchinnikov DA, Sun J, Fortuna PR, Wolvetang EJ. (2014). Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev, 23(13), 1515-1523. https://doi.org/10.1089/scd.2013.0461

Publication

Stem cells and development

ISSN: 1557-8534

NlmUniqueID: 101197107

Country: United States

Language: English

Volume: 23

Issue: 13

Pages: 1515-1523

Researcher Affiliations

Whitworth, Deanne J

1 School of Veterinary Science, University of Queensland , Gatton, Queensland, Australia .

Ovchinnikov, Dmitry A

Sun, Jane

Fortuna, Patrick R J

Wolvetang, Ernst J

MeSH Terms

Animals
Biomarkers / metabolism
Cell Proliferation
Coculture Techniques
Feeder Cells
Female
Fibroblast Growth Factors / physiology
Fibroblasts / physiology
Gene Expression
Histones / metabolism
Horses
Induced Pluripotent Stem Cells / physiology
Leukemia Inhibitory Factor / physiology
Skin / cytology
Transcription Factors / metabolism
X Chromosome / genetics

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