Efficient method for generating nuclear fractions from marrow stromal cells

Abstract

Stem cells have received significant attention for their envisioned potential to treat currently unapproachable diseases. No less important is the utility of stem cells to serve as model systems of differentiation. Analyses at the transcriptome, miRNA and proteome levels have yielded valuable insights into events underlying stem cell differentiation. Proteomic analysis is often cumbersome, detecting changes in hundreds of proteins that require subsequent identification and validation. Targeted analysis of nuclear constituents would simplify proteomic studies, focusing efforts on transcription factor abundance and modification. To facilitate such studies, a simple and efficient methodology to isolate pure nuclear fractions from Marrow Stromal Cells (MSCs), a clinically relevant stem cell population, has been developed. The modified protocol greatly enhances cell disruption, yielding free nuclei without attached cell body remnants. Light and electron microscopic analysis of purified nuclei demonstrated that preparations contained predominantly intact nuclei with minimal cytoplasmic contamination. Western analysis revealed an approximately eightfold enrichment of the transcription factor CREB in the isolated nuclei over that in the starting homogenates. This simple method for isolation of highly purified nuclear fractions from stem cell populations will allow rigorous examination of nuclear proteins critical for differentiation.