Mechanical phenotyping of primary human skeletal stem cells in heterogeneous populations by real-time deformability cytometry

Abstract

Skeletal stem cells (SSCs) are a sub-population of mesenchymal stromal cells (MSCs) present in bone marrow with multipotent differentiation potential. A current unmet challenge hampering their clinical translation remains the isolation of homogeneous populations of SSCs, in vitro, with consistent regeneration and differentiation capacities. Cell stiffness has been shown to play an important role in cell separation using microfluidic techniques such as inertial focusing or deterministic lateral displacement. Here we report that the mechanical properties of SSCs, and of a surrogate human osteosarcoma cell line (MG-63), differ significantly from other cell populations found in the bone marrow. Using real-time deformability cytometry, a recently introduced method for cell mechanical characterization, we demonstrate that both MG-63 and SSCs are stiffer than the three primary leukocyte lineages (lymphocytes, monocytes and granulocytes) and also stiffer than HL-60, a human leukemic progenitor cell line. In addition, we show that SSCs form a mechanically distinct sub-population of MSCs. These results represent an important step towards finding the bio-physical fingerprint of human SSCs that will allow their label-free separation from bone marrow with significant physiological and therapeutic implications.

Statistics for real-time deformability cytometry: Clustering, dimensionality reduction, and significance testing

“…Dataset and methods for cell preparation have been published earlier 23 . Briefly, the MG-63 human osteosarcoma cell line was cultured in Dulbecco Modified Eagle Medium with 10% fetal calf serum (Lonza, Basel, Switzerland), 100 U/ml penicillin, and 100  μ g/ml streptomycin at 37 °C.…”

“…Dataset and methods for cell preparation have been published earlier 23 . Briefly, the human skeletal stem cells were obtained during total hip replacement at Southampton General Hospital or the Spire Southampton Hospital in accordance with the Southampton and South West Hampshire Research Ethics Committee (Ref.…”

“…Gaussian mixture models (GMM) are implemented to identify a sub-population in mesenchymal stem cells based on biophysical properties 23 . Additionally, we show that a simple dimensionality reduction using principal component analysis (PCA) is sufficient to discover a small very distinct sub-population in a retina sample, only using morphological and rheological information.…”

“…In an exemplary use case, we show differences in mechanical properties between the human osteosarcoma cell line MG-63 and skeletal stem cells (SSCs) and discuss biological implications for an improved understanding of mesenchymal stroma cells 23 . The statistical test was implemented into an analysis software for RT-DC data with a graphical user interface 36 …”

Skeletal stem cell isolation: A review on the state-of-the-art microfluidic label-free sorting techniques

“…High-throughput single-cell characterisation techniques like microfluidic impedance cytometry (Sun and Morgan, 2010) and real-time deformability cytometry (RT-DC) (Otto et al, 2015) can provide data on cell dielectric and elastic properties. Using RT-DC it has recently been shown, that enriched SSC populations, are significantly stiffer than cells from the haematopoietic lineage found in the bone marrow, namely, lymphocytes, monocytes, granulocytes and a blood progenitor cell line, HL-60, which are commonly used as a model for HSCs (Xavier et al, 2016). Such differences could be exploited for separation using for example DLD, which is sensitive to differences in cells deformability (Beech et al, 2012;Holmes et al, 2014).…”