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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.


Cellular Morphology. Light microscopic observation of confluent hESCs after 72 hours maintained in PPP-derived hydrogel and on fibronectin. Scale bar represents 200 μm.
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Fig2: Cellular Morphology. Light microscopic observation of confluent hESCs after 72 hours maintained in PPP-derived hydrogel and on fibronectin. Scale bar represents 200 μm.

Mentions: Undifferentiated hESCs typically exhibit a high nucleus to cytoplasm ratio and form colonies. hESCs embedded within the hydrogel and cultured using DMEM/F12 as detailed previously were observed to proliferate effectively in vitro and had successfully formed colonies displaying typical hESC morphology 24 hours following their seeding within the hydrogel. Expansion of the hESC populations continued until reaching confluency after 72 hours as illustrated in figure 2.Figure 2


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)

Cellular Morphology. Light microscopic observation of confluent hESCs after 72 hours maintained in PPP-derived hydrogel and on fibronectin. Scale bar represents 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Cellular Morphology. Light microscopic observation of confluent hESCs after 72 hours maintained in PPP-derived hydrogel and on fibronectin. Scale bar represents 200 μm.
Mentions: Undifferentiated hESCs typically exhibit a high nucleus to cytoplasm ratio and form colonies. hESCs embedded within the hydrogel and cultured using DMEM/F12 as detailed previously were observed to proliferate effectively in vitro and had successfully formed colonies displaying typical hESC morphology 24 hours following their seeding within the hydrogel. Expansion of the hESC populations continued until reaching confluency after 72 hours as illustrated in figure 2.Figure 2

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.