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Defining synthetic surfaces for human pluripotent stem cell culture.

Lambshead JW, Meagher L, O'Brien C, Laslett AL - Cell Regen (Lond) (2013)

Bottom Line: Human pluripotent stem cells (hPSCs) are able to self-renew indefinitely and to differentiate into all adult cell types. hPSCs therefore show potential for application to drug screening, disease modelling and cellular therapies.Most hPSC culture surfaces have been derived from extracellular matrix proteins (ECMPs) and their cell adhesion molecule (CAM) binding motifs.Reports indicate that hPSC cultures can be supported by cell-surface interactions through certain CAM subtypes but not by others.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Materials Science and Engineering, Clayton, Victoria 3168 Australia ; Australian Regenerative Medicine Institute, Monash University, Kragujevac, Victoria 3800 Australia.

ABSTRACT
Human pluripotent stem cells (hPSCs) are able to self-renew indefinitely and to differentiate into all adult cell types. hPSCs therefore show potential for application to drug screening, disease modelling and cellular therapies. In order to meet this potential, culture conditions must be developed that are consistent, defined, scalable, free of animal products and that facilitate stable self-renewal of hPSCs. Several culture surfaces have recently been reported to meet many of these criteria although none of them have been widely implemented by the stem cell community due to issues with validation, reliability and expense. Most hPSC culture surfaces have been derived from extracellular matrix proteins (ECMPs) and their cell adhesion molecule (CAM) binding motifs. Elucidating the CAM-mediated cell-surface interactions that are essential for the in vitro maintenance of pluripotency will facilitate the optimisation of hPSC culture surfaces. Reports indicate that hPSC cultures can be supported by cell-surface interactions through certain CAM subtypes but not by others. This review summarises the recent reports of defined surfaces for hPSC culture and focuses on the CAMs and ECMPs involved.

No MeSH data available.


Related in: MedlinePlus

HPSCs cultured on different surfaces. Schematic diagrams illustrate the arrangement of cell adhesion molecules (CAMs), ligands and substrates (where appropriate) of the three major types of culture surfaces used for maintenance of human pluripotent stem cells (hPSCs). (A) Feeder cells, (B) extracellular matrix (ECM) extracts and (C) chemically defined culture surfaces. (D-F) Phase contrast images of hPSCs cultured on one example of each surface type, murine embryonic fibroblasts, GeltrexTM and Corning SynthemaxTM respectively. (D*-F*) Magnified regions of D-F. hPSCs cultured on each surface display a typical morphology with compact colonies of cells with prominent nucleoli and high nuclear-to-cytoplasmic ratio.
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Fig2: HPSCs cultured on different surfaces. Schematic diagrams illustrate the arrangement of cell adhesion molecules (CAMs), ligands and substrates (where appropriate) of the three major types of culture surfaces used for maintenance of human pluripotent stem cells (hPSCs). (A) Feeder cells, (B) extracellular matrix (ECM) extracts and (C) chemically defined culture surfaces. (D-F) Phase contrast images of hPSCs cultured on one example of each surface type, murine embryonic fibroblasts, GeltrexTM and Corning SynthemaxTM respectively. (D*-F*) Magnified regions of D-F. hPSCs cultured on each surface display a typical morphology with compact colonies of cells with prominent nucleoli and high nuclear-to-cytoplasmic ratio.

Mentions: The quality of ongoing hPSC cultures should be regularly assessed. When developing or implementing novel culture conditions it is important to characterise the cells thoroughly in order to validate the culture system. Daily assessment of hPSC cultures should involve visual observation of characteristic tightly-packed colonies of cuboidal-shaped cells containing prominent nuclei, multiple nucleoli and little cytoplasm, with minimal differentiated cell types present as shown in Figure 2[1]. Proliferation rates of ongoing cultures can be monitored over time by recording approximate cell seeding densities and the frequency of passaging, but when comparing various culture conditions the proliferation rate should be calculated more accurately from serial cell counts of ongoing cultures at multiple time points. Stronger evidence for pluripotency can be generated by monitoring associated molecular markers. The gold standard genetic marker of pluripotency is POU domain, class 5, transcription factor 1 (Pou5f1) aka OCT4, a homeodomain transcription factor of the POU family that is essential for pluripotent cells [24]. Expression of OCT4 and other markers can be assessed in populations of hPSCs using numerous methods, listed in Table 1[25–28]. Additional information about the cell state can be obtained by characterising the epigenetic signature. Epigenetic regulation of gene expression is exercised through modifications to the genome that do not affect the genetic sequence. DNA methylation is one of the most-studied epigenetic modifications. Methylation down-regulates expression of local genes and can be detected by sequencing bisulfite-treated DNA [29]. Signature methylation patterns can be used to identify developmentally regulated cell types and individual hPSC lines and change in response to environmental stimuli [reviewed by [30]. DNA methylation patterns have also been linked to the differentiation potential of hPSCs and can therefore be used as molecular markers of pluripotency [31]. Molecular markers are however not completely specific to pluripotent cells due to the inherent heterogeneity of hPSCs. For example subpopulations with reduced differentiation potential have been identified within OCT4-positive populations of hPSCs [21]. While combinatorial assessment of marker expression improves the robustness of molecular assays for pluripotency they ultimately remain surrogate assays, whereas functional demonstrations of cell potential provide more stringent tests of pluripotency. The ability of hPSCs to differentiate into cell types of all three embryonic germ layers (endoderm, ectoderm and mesoderm) can be examined both in vitro and in vivo. In vitro differentiation of pluripotent cells is usually associated with the formation of embryoid bodies [complex, non-adherent, three-dimensional structures composed of spontaneously differentiating hPSCs [32, 33] and can either be spontaneous or directed towards certain cell fates [25, 34]. The in vivo differentiation potential of hPSCs is typically tested by transplantation into immunodeficient mice. The formation of a teratoma (a benign tumour comprising cell types representative of each of the three embryonic germ layers) at the site of implantation is the most stringent validation assay available for the differentiation potential of putative hPSCs [18]. However, differentiation assays are laborious, inconsistent in efficiency and difficult to standardise across cell lines and laboratories, so evaluation of molecular markers remains important for assessing the efficacy of hPSC culture systems. Quality control of any long-term cell culture system should also include an assessment of genetic stability using G-banding analysis to detect gross or subchromosomal changes. However, genetic aberrations below the detection limit of G-banding have been identified in hPSC lines and more detailed genetic analysis should also be considered when testing novel culture systems [35]. A detailed characterisation of hPSCs should include the methodologies bolded in Table 1.Figure 2


Defining synthetic surfaces for human pluripotent stem cell culture.

Lambshead JW, Meagher L, O'Brien C, Laslett AL - Cell Regen (Lond) (2013)

HPSCs cultured on different surfaces. Schematic diagrams illustrate the arrangement of cell adhesion molecules (CAMs), ligands and substrates (where appropriate) of the three major types of culture surfaces used for maintenance of human pluripotent stem cells (hPSCs). (A) Feeder cells, (B) extracellular matrix (ECM) extracts and (C) chemically defined culture surfaces. (D-F) Phase contrast images of hPSCs cultured on one example of each surface type, murine embryonic fibroblasts, GeltrexTM and Corning SynthemaxTM respectively. (D*-F*) Magnified regions of D-F. hPSCs cultured on each surface display a typical morphology with compact colonies of cells with prominent nucleoli and high nuclear-to-cytoplasmic ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230363&req=5

Fig2: HPSCs cultured on different surfaces. Schematic diagrams illustrate the arrangement of cell adhesion molecules (CAMs), ligands and substrates (where appropriate) of the three major types of culture surfaces used for maintenance of human pluripotent stem cells (hPSCs). (A) Feeder cells, (B) extracellular matrix (ECM) extracts and (C) chemically defined culture surfaces. (D-F) Phase contrast images of hPSCs cultured on one example of each surface type, murine embryonic fibroblasts, GeltrexTM and Corning SynthemaxTM respectively. (D*-F*) Magnified regions of D-F. hPSCs cultured on each surface display a typical morphology with compact colonies of cells with prominent nucleoli and high nuclear-to-cytoplasmic ratio.
Mentions: The quality of ongoing hPSC cultures should be regularly assessed. When developing or implementing novel culture conditions it is important to characterise the cells thoroughly in order to validate the culture system. Daily assessment of hPSC cultures should involve visual observation of characteristic tightly-packed colonies of cuboidal-shaped cells containing prominent nuclei, multiple nucleoli and little cytoplasm, with minimal differentiated cell types present as shown in Figure 2[1]. Proliferation rates of ongoing cultures can be monitored over time by recording approximate cell seeding densities and the frequency of passaging, but when comparing various culture conditions the proliferation rate should be calculated more accurately from serial cell counts of ongoing cultures at multiple time points. Stronger evidence for pluripotency can be generated by monitoring associated molecular markers. The gold standard genetic marker of pluripotency is POU domain, class 5, transcription factor 1 (Pou5f1) aka OCT4, a homeodomain transcription factor of the POU family that is essential for pluripotent cells [24]. Expression of OCT4 and other markers can be assessed in populations of hPSCs using numerous methods, listed in Table 1[25–28]. Additional information about the cell state can be obtained by characterising the epigenetic signature. Epigenetic regulation of gene expression is exercised through modifications to the genome that do not affect the genetic sequence. DNA methylation is one of the most-studied epigenetic modifications. Methylation down-regulates expression of local genes and can be detected by sequencing bisulfite-treated DNA [29]. Signature methylation patterns can be used to identify developmentally regulated cell types and individual hPSC lines and change in response to environmental stimuli [reviewed by [30]. DNA methylation patterns have also been linked to the differentiation potential of hPSCs and can therefore be used as molecular markers of pluripotency [31]. Molecular markers are however not completely specific to pluripotent cells due to the inherent heterogeneity of hPSCs. For example subpopulations with reduced differentiation potential have been identified within OCT4-positive populations of hPSCs [21]. While combinatorial assessment of marker expression improves the robustness of molecular assays for pluripotency they ultimately remain surrogate assays, whereas functional demonstrations of cell potential provide more stringent tests of pluripotency. The ability of hPSCs to differentiate into cell types of all three embryonic germ layers (endoderm, ectoderm and mesoderm) can be examined both in vitro and in vivo. In vitro differentiation of pluripotent cells is usually associated with the formation of embryoid bodies [complex, non-adherent, three-dimensional structures composed of spontaneously differentiating hPSCs [32, 33] and can either be spontaneous or directed towards certain cell fates [25, 34]. The in vivo differentiation potential of hPSCs is typically tested by transplantation into immunodeficient mice. The formation of a teratoma (a benign tumour comprising cell types representative of each of the three embryonic germ layers) at the site of implantation is the most stringent validation assay available for the differentiation potential of putative hPSCs [18]. However, differentiation assays are laborious, inconsistent in efficiency and difficult to standardise across cell lines and laboratories, so evaluation of molecular markers remains important for assessing the efficacy of hPSC culture systems. Quality control of any long-term cell culture system should also include an assessment of genetic stability using G-banding analysis to detect gross or subchromosomal changes. However, genetic aberrations below the detection limit of G-banding have been identified in hPSC lines and more detailed genetic analysis should also be considered when testing novel culture systems [35]. A detailed characterisation of hPSCs should include the methodologies bolded in Table 1.Figure 2

Bottom Line: Human pluripotent stem cells (hPSCs) are able to self-renew indefinitely and to differentiate into all adult cell types. hPSCs therefore show potential for application to drug screening, disease modelling and cellular therapies.Most hPSC culture surfaces have been derived from extracellular matrix proteins (ECMPs) and their cell adhesion molecule (CAM) binding motifs.Reports indicate that hPSC cultures can be supported by cell-surface interactions through certain CAM subtypes but not by others.

View Article: PubMed Central - PubMed

Affiliation: CSIRO Materials Science and Engineering, Clayton, Victoria 3168 Australia ; Australian Regenerative Medicine Institute, Monash University, Kragujevac, Victoria 3800 Australia.

ABSTRACT
Human pluripotent stem cells (hPSCs) are able to self-renew indefinitely and to differentiate into all adult cell types. hPSCs therefore show potential for application to drug screening, disease modelling and cellular therapies. In order to meet this potential, culture conditions must be developed that are consistent, defined, scalable, free of animal products and that facilitate stable self-renewal of hPSCs. Several culture surfaces have recently been reported to meet many of these criteria although none of them have been widely implemented by the stem cell community due to issues with validation, reliability and expense. Most hPSC culture surfaces have been derived from extracellular matrix proteins (ECMPs) and their cell adhesion molecule (CAM) binding motifs. Elucidating the CAM-mediated cell-surface interactions that are essential for the in vitro maintenance of pluripotency will facilitate the optimisation of hPSC culture surfaces. Reports indicate that hPSC cultures can be supported by cell-surface interactions through certain CAM subtypes but not by others. This review summarises the recent reports of defined surfaces for hPSC culture and focuses on the CAMs and ECMPs involved.

No MeSH data available.


Related in: MedlinePlus