Center of Biomedical Research Excellence (COBRE) for Skeletal Health and Repair

High-yield, Lineage-specific Enrichment of Living Mesenchymal Stem Cells

Principal Investigator: Eric Darling, PhD


Mesenchymal stem cells pose exciting possibilities for cell-based regenerative therapies. Their capability to differentiate along multiple musculoskeletal lineages makes them attractive as an alternative source for autologous cells. However, these cells must first be isolated from other cell types before use in cell therapies, and they have proven to be difficult to enrich using antibody-based sorting approaches. Typical yields are less than 1-percent, which results in insufficient cell numbers for single-surgery clinical procedures. Alternative enrichment strategies are needed that can identify large numbers of cells by lineage potential, rather than relying only on surface markers. In this project, we will investigate mechanical- and gene expression- based approaches for osteogenic and adipogenic lineage enrichment. Mechanical properties will be assessed using atomic force microscopy, a technique that allows highly controlled force measurements at small scales.

To accomplish the overall goals of the project we will:
  • Specific Aim 1: Determine whether mechanical biomarkers are indicative of lineage potential. The proposed mechanical tests will evaluate cellular mechanical properties before, during and after osteogenic and adipogenic differentiation.
  • Specific Aim 2: Develop a live-cell molecular beacon assay that distinguishes differentiating cells from non-differentiating cells based on lineage-specific gene expressions. The proposed beacons will target early and late stage markers for osteogenesis and adipogenesis.
  • Specific Aim 3: Validate the use of mechanical and molecular beacon markers for enrichment of lineage-specific cells. The proposed experiment will evaluate the osteogenic and adipogenic capabilities of sorted cells from lipoaspirate.

Adult stem cells are a promising option for repairing tissues damaged by injury or disease. To be effective, the purity of the stem cell population needs to be sufficiently high, a task that has yet to be solved. The goal of this research is to evaluate whether cellular mechanical properties and lineage-specific gene expressions can be used to enrich for cells capable of regenerating either bone or fat tissues.

  1. Gonzalez-Cruz RD, Fonseca VC, Darling EM. 2012. Cellular mechanical properties reflect differentiation potential of adipose-derived mesenchymal stem cells. Proc Natl Acad Sci USA. 109 (24) E1523-9. PMCID: PMC3386052
  2. Desai HV, Voruganti IS, Jayasuriya C, Chen Q, Darling EM. 2013. Live-cell, temporal gene expression analysis of osteogenic differentiation in adipose-derived stem cells. Tissue Eng A. 19 (1-2) pp. 40-48. PMCID: PMC3530940
  3. Gonzalez-Cruz RD, Darling EM. 2013. Adipose-derived stem cell fate is predicted by cellular mechanical properties. Adipocyte. 2 (2) pp. 1-5.