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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

Teratoma Formation. Teratoma formation in immunodeficient mice demonstrates differentiation into derivatives of the three germ layers. A: Macroscopic observation of teratoma formation. B: Glandular structures (endoderm). C: Muscle (mesoderm), adipose (mesoderm), hair follicles (ectoderm). D: Blood vessels (mesoderm). Scale bar represents 100 μm.
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Fig6: Teratoma Formation. Teratoma formation in immunodeficient mice demonstrates differentiation into derivatives of the three germ layers. A: Macroscopic observation of teratoma formation. B: Glandular structures (endoderm). C: Muscle (mesoderm), adipose (mesoderm), hair follicles (ectoderm). D: Blood vessels (mesoderm). Scale bar represents 100 μm.

Mentions: Implantation of hESCs maintained up to passage 25 led to the formation of teratomas 6 weeks post-injection observed both macroscopically and microscopically in each case, n = 6. Derivatives of all three germ layers were identified including endoderm-derived glandular structures, ectoderm –derived hair follicles and a variety of mesoderm-derived structures including adipocytes, myotubes and blood vessels. This provided further confirmation that hESCs maintain their differentiation potential when maintained using this novel PPP-derived hydrogel culture system (figure 6).Figure 6


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)

Teratoma Formation. Teratoma formation in immunodeficient mice demonstrates differentiation into derivatives of the three germ layers. A: Macroscopic observation of teratoma formation. B: Glandular structures (endoderm). C: Muscle (mesoderm), adipose (mesoderm), hair follicles (ectoderm). D: Blood vessels (mesoderm). Scale bar represents 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Teratoma Formation. Teratoma formation in immunodeficient mice demonstrates differentiation into derivatives of the three germ layers. A: Macroscopic observation of teratoma formation. B: Glandular structures (endoderm). C: Muscle (mesoderm), adipose (mesoderm), hair follicles (ectoderm). D: Blood vessels (mesoderm). Scale bar represents 100 μm.
Mentions: Implantation of hESCs maintained up to passage 25 led to the formation of teratomas 6 weeks post-injection observed both macroscopically and microscopically in each case, n = 6. Derivatives of all three germ layers were identified including endoderm-derived glandular structures, ectoderm –derived hair follicles and a variety of mesoderm-derived structures including adipocytes, myotubes and blood vessels. This provided further confirmation that hESCs maintain their differentiation potential when maintained using this novel PPP-derived hydrogel culture system (figure 6).Figure 6

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