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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.


HESC Viability. Single field in which live/dead staining of hESCs embedded within PPP-derived hydrogel is shown after 72 hours in culture. A: Live cells (green). B: Dead cells (red). C: Nuclei (blue).
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Fig1: HESC Viability. Single field in which live/dead staining of hESCs embedded within PPP-derived hydrogel is shown after 72 hours in culture. A: Live cells (green). B: Dead cells (red). C: Nuclei (blue).

Mentions: Subsequent viability tests confirmed that hESCs cultured within the PPP-derived hydrogel remained viable (green fluorescence) over 72 hours in vitro. Although occasionally cells fluorescing red were identified, indicating the binding of ethidium bromide to the DNA of cells with damaged membranes, this was rare and did not indicate any unusual loss of cell viability (figure 1).Figure 1


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)

HESC Viability. Single field in which live/dead staining of hESCs embedded within PPP-derived hydrogel is shown after 72 hours in culture. A: Live cells (green). B: Dead cells (red). C: Nuclei (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: HESC Viability. Single field in which live/dead staining of hESCs embedded within PPP-derived hydrogel is shown after 72 hours in culture. A: Live cells (green). B: Dead cells (red). C: Nuclei (blue).
Mentions: Subsequent viability tests confirmed that hESCs cultured within the PPP-derived hydrogel remained viable (green fluorescence) over 72 hours in vitro. Although occasionally cells fluorescing red were identified, indicating the binding of ethidium bromide to the DNA of cells with damaged membranes, this was rare and did not indicate any unusual loss of cell viability (figure 1).Figure 1

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.