Unknown,Transcriptomics,Genomics,Proteomics

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Hybridisation of 11 8-cell neonatal foreskin keratinocyte colonies

ABSTRACT: Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms which enable this are ill defined. Using live imaging we saw two populations of cells those which generate expanding colonies and those which generate balanced colonies. We wanted to assess if there were any transcriptional differences between these two colony types.

ORGANISM(S): Homo sapiens

SUBMITTER: Jo Fowler

PROVIDER: E-MTAB-3128 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Human multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible. Cells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation. IN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30-75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded. The results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities. Experiment Overall Design: In this study heterogeneity of human multipotent stromal cells in single-cell-derived colonies was examined. Cells were grown as colonies on slides for 12 days and cells from inside (IN) and outside (OUT) of colonies were isolated with laser capture microdissection. Cells from two colonies were pooled for each sample. This study consists of 3 biological replicates for each colony region (3 for IN and 3 for OUT). All 6 samples were run on Affymetrix microarrays after amplification and labeling using Nugen Ovation technology.

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Hybridisation of 11 8-cell neonatal foreskin keratinocyte colonies

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