Limits...
A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro.

Lewis FC, Bryan N, Hunt JA - Cell Regen (Lond) (2012)

Bottom Line: Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively.PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ.Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Clinical Engineering, UKCTE, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, L69 3GA UK.

ABSTRACT

Background: Human embryonic stem cells (hESCs) represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF) feeder-layers and exogenous protein media supplementation.

Results: These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP), which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages.

Conclusions: PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus

Pluripotency Gene Expression. Quantification of pluripotency gene expression after culture under PPP-derived hydrogel and fibronectin culture conditions at passage 1, 5 and 10. Error bars represent 1 standard deviation from the mean, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230398&req=5

Fig4: Pluripotency Gene Expression. Quantification of pluripotency gene expression after culture under PPP-derived hydrogel and fibronectin culture conditions at passage 1, 5 and 10. Error bars represent 1 standard deviation from the mean, n = 3.

Mentions: Over the period of culture, expression of OCT4, NANOG and SOX2 was comparable between both hESCs maintained using both PPP-hydrogel and fibronectin conditions over a number of passages with no significant difference in transcript expression (figure 4).Figure 4


A feeder-free, human plasma-derived hydrogel for maintenance of a human embryonic stem cell phenotype in vitro.

Lewis FC, Bryan N, Hunt JA - Cell Regen (Lond) (2012)

Pluripotency Gene Expression. Quantification of pluripotency gene expression after culture under PPP-derived hydrogel and fibronectin culture conditions at passage 1, 5 and 10. Error bars represent 1 standard deviation from the mean, n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230398&req=5

Fig4: Pluripotency Gene Expression. Quantification of pluripotency gene expression after culture under PPP-derived hydrogel and fibronectin culture conditions at passage 1, 5 and 10. Error bars represent 1 standard deviation from the mean, n = 3.
Mentions: Over the period of culture, expression of OCT4, NANOG and SOX2 was comparable between both hESCs maintained using both PPP-hydrogel and fibronectin conditions over a number of passages with no significant difference in transcript expression (figure 4).Figure 4

Bottom Line: Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively.PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ.Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Clinical Engineering, UKCTE, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, L69 3GA UK.

ABSTRACT

Background: Human embryonic stem cells (hESCs) represent a tremendous resource for cell therapies and the study of human development; however to maintain their undifferentiated state in vitro they routinely require the use of mouse embryonic fibroblast (MEF) feeder-layers and exogenous protein media supplementation.

Results: These well established requirements can be overcome and in this study, it will be demonstrated that phenotypic stability of hESCs can be maintained using a novel, human plasma protein-based hydrogel as an extracellular culture matrix without the use of feeder cell co-culture. hESCs were resuspended in human platelet poor plasma (PPP), which was gelled by the addition of calcium containing DMEM-based hESC culture medium. Phenotypic and genomic expression of the pluripotency markers OCT4, NANOG and SOX2 were measured using immunohistochemistry and qRT-PCR respectively. Typical hESC morphology was demonstrated throughout in vitro culture and both viability and phenotypic stability were maintained throughout extended culture, up to 25 passages.

Conclusions: PPP-derived hydrogel has demonstrated to be an efficacious alternative to MEF co-culture with its hydrophilicity allowing for this substrate to be delivered via minimally invasive procedures in a liquid phase with polymerization ensuing in situ. Together this provides a novel technique for the study of this unique group of stem cells in either 2D or 3D both in vitro and in vivo.

No MeSH data available.


Related in: MedlinePlus