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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.


Related in: MedlinePlus

Identification of Pluripotency Markers. Fluorescent observation of passage 25 hESCs immunohistochemically stained for OCT4, NANOG, SOX2 (left), DAPI (centre), merged (right) under PPP-derived hydrogel conditions. Scale bar represents 50 μm.
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Fig3: Identification of Pluripotency Markers. Fluorescent observation of passage 25 hESCs immunohistochemically stained for OCT4, NANOG, SOX2 (left), DAPI (centre), merged (right) under PPP-derived hydrogel conditions. Scale bar represents 50 μm.

Mentions: Morphological analysis of hESCs embedded within the hydrogel suggested that hESCs remained undifferentiated. To confirm this observation, immunohistochemistry was performed on the seeded PPP-derived hydrogels to identify pluripotency markers characteristic of hESC undifferentiated phenotype: the transcription factors OCT4NANOG and SOX2, all of which are typically expressed by undifferentiated hESCs in standard culture [12–14] along with the surface antigen SSEA4 were highlighted as indicators of a pluripotent phenotype. All nuclear transcription factors were clearly identified up to 25 passages as illustrated in figure 3.Figure 3


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)

Identification of Pluripotency Markers. Fluorescent observation of passage 25 hESCs immunohistochemically stained for OCT4, NANOG, SOX2 (left), DAPI (centre), merged (right) under PPP-derived hydrogel conditions. Scale bar represents 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Identification of Pluripotency Markers. Fluorescent observation of passage 25 hESCs immunohistochemically stained for OCT4, NANOG, SOX2 (left), DAPI (centre), merged (right) under PPP-derived hydrogel conditions. Scale bar represents 50 μm.
Mentions: Morphological analysis of hESCs embedded within the hydrogel suggested that hESCs remained undifferentiated. To confirm this observation, immunohistochemistry was performed on the seeded PPP-derived hydrogels to identify pluripotency markers characteristic of hESC undifferentiated phenotype: the transcription factors OCT4NANOG and SOX2, all of which are typically expressed by undifferentiated hESCs in standard culture [12–14] along with the surface antigen SSEA4 were highlighted as indicators of a pluripotent phenotype. All nuclear transcription factors were clearly identified up to 25 passages as illustrated in figure 3.Figure 3

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