Abstract
Human Multipotent Stromal Cells (MSCs) are a valuable resource for regenerative medicine and are widely studied. They can be isolated from a variety of tissues and differentiate into multiple cell types (multi-potent). Many reports have been published using human MSCs and to be able to compare outcome, or be able to identify differences between MSCs, several cell surface markers have been proposed. Nevertheless, still many differences remain. Gene expression is known to be different between cell stage and origin. Furthermore, cells cultured on a culture dish (2D) show different gene expression profiles as compared to cells grown on scaffolds (3D). Even the RNA extraction method and the selection of genes used for normalisation have a role in gene expression profiling. To be able to compare gene expression data from samples cultured in different dimensions and RNA extracted using a variety of protocols we set out to define a set of reference genes suitable to normalise qPCR data from a very heterogeneous sample set. Hereto, Trizol was used to extract RNA from human MSCs cultured in 3D and 2D to validate newly designed and previously published primer sets. Subsequently, RNA from fresh human MSC samples and samples stored in RLT-buffer, Trizol or RNAlater was extracted using RNeasy and Trizol methods. All samples have been used to rank the candidate reference genes according to their stability after qPCR enabling identification of the most suitable reference gene(s) for normalisation of a heterogeneous sample set. The most stably expressed reference genes indicated superior normalisation of MSC marker gene expression over the least stable reference genes.
Highlights
Multipotent stromal cells, known as mesenchymal stem cells, mesenchymal stromal cells or medicinal signalling cells [1] (MSCs), hold great promise for regenerative medicine and tissue engineering
Multipotent Stromal Cells (MSCs), immortalized by transduction with human telomerase reverse transcriptase, referred to as MSC-hTERT [15] and MSCs derived from umbilical cord (UC) were cultured to obtain a variety of RNA samples
RNA samples from MSC-hTERT and UC derived MSCs (UC-MSCs) cultures were obtained using different extraction protocols (RNeasy and Trizol) after cell storage in different media (RLT, Trizol, and RNAlater) for several time periods (0 days, 7 days, and 30 days) to identify candidate reference gene (RG) for accurate normalisation of gene expression in human MSCs cultured in 2D and 3D (Table 2 and Fig 2A)
Summary
Multipotent stromal cells, known as mesenchymal stem cells, mesenchymal stromal cells or medicinal signalling cells [1] (MSCs), hold great promise for regenerative medicine and tissue engineering. Reference genes for hMSC qPCR and regenerative capacity through secretion of bioactive macromolecules [3]. Because of their relatively easy isolation from many tissues the identity of the true MSC is unknown. The definition of the MSC phenotype is focussed on the expression of cellular surface markers (CD105, CD90 and CD73) or the lack thereof (e.g. CD45, CD34, CD19 and CD11b) [4]. These minimal requirements resulted in numerous reports using MSCs with variable outcome regarding maintaining multipotency or differentiation potency and efficiency. Other reasons why differences between MSCs can be observed are the age of the donor [5] and the duration of culture of the MSCs [6]
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