Identification of stable reference genes in differentiating human pluripotent stem cells

Area of Science:

• Stem Cell Biology
• Molecular Biology
• Genomics

Background:

• Housekeeping genes (HKGs) are assumed stable for normalizing gene expression.
• Previous studies and our prior work indicate HKG variability in human embryonic stem cells (hESCs).
• This variability challenges accurate gene expression analysis in stem cell research.

Purpose of the Study:

• To investigate the expression stability of HKGs in human pluripotent stem cells (hPSCs) across various differentiation conditions.
• To identify reliable reference genes for normalizing gene expression data in hPSCs.
• To establish a distinct HKG signature for differentiating hPSCs.

Main Methods:

• Analysis of nine global gene expression datasets from hESCs and human induced pluripotent stem cells (hiPSCs).
• Examination of gene expression during directed differentiation towards endoderm, mesoderm, and ectoderm derivatives.
• Validation of identified stable genes using reverse transcription quantitative PCR (RT-qPCR).

Main Results:

• Commonly used HKGs exhibited significant expression variability in differentiating hPSCs.
• A set of stably expressed genes, including EID2, ZNF324B, CAPN10, and RABEP2, were identified as suitable reference genes for hPSCs.
• These novel reference genes demonstrated consistent expression across different cell lines and experimental conditions.

Conclusions:

• Differentiating hPSCs possess a unique HKG signature that differs from somatic cells.
• Validation of reference gene stability is crucial for experimental setups involving hPSCs.
• The identified novel HKGs provide a more reliable option for normalizing gene expression data in differentiating hPSCs.