Monitoring live human mesenchymal stromal cell differentiation and subsequent selection using fluorescent RNA-based probes

Sci Rep. 2016 May 20:6:26014. doi: 10.1038/srep26014.

Abstract

Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bone Marrow Cells / physiology
  • Bone Regeneration
  • Cell Differentiation
  • Cell Separation
  • Cells, Cultured
  • Core Binding Factor Alpha 1 Subunit / genetics*
  • Flow Cytometry
  • Fluorescent Dyes
  • Humans
  • Mesenchymal Stem Cells / physiology*
  • Osteocalcin / metabolism
  • Osteogenesis
  • RNA Probes / genetics
  • RNA, Messenger / genetics*
  • SOX9 Transcription Factor / genetics*
  • Single-Cell Analysis / methods*
  • Tissue Engineering

Substances

  • Core Binding Factor Alpha 1 Subunit
  • Fluorescent Dyes
  • RNA Probes
  • RNA, Messenger
  • RUNX2 protein, human
  • SOX9 Transcription Factor
  • SOX9 protein, human
  • Osteocalcin