Dataset Information

Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells

ABSTRACT: Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional-culture hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. The AIC medium shows a good compatibility for suspension expansion of different hPSC lines. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products

ORGANISM(S): Homo sapiens

PROVIDER: GSE145163 | GEO | 2020/04/23

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them good sources of cells for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a systematic study over more than 100continuous passages to identify characteristics of culture conditions (including passage method, substrate, and media type) that influence the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. The predominant effects we observed were increased genetic instability with enzymatic passage, higher cell proliferation with feeder-free substrate, and variations among cultures in global gene expression and DNA methylation with time in culture. We observed recurrent duplications in two genomic regions that have been noted in earlier studies to be hotspots for duplication in hPSCs, as well as a previously unreported recurrent deletion of the tumor suppressor gene TP53 in all but one of the long-term culture conditions; the exception was the condition using mechanical passaging on feeder layers. The deletion of TP53 is associated with decreased mRNA expression of TP53, as well as alterations in the expression of several other genes in the TP53 pathway, which taken together indicate a decrease in the function of the TP53 pathway. Our results highlight the need for careful assessment of effects of culture conditions on cells intended for clinical therapies. Total RNA extracted at different passages from Human Embryonic Stem Cells in different culture conditions. Total DNA extracted at different passages from Human Embryonic Stem Cells in different culture conditions.

Dataset Information

Modulation of Wnt and Activin/Nodal supports efficient derivation, cloning and suspension expansion of human pluripotent stem cells

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure