Size and dielectric properties of skeletal stem cells change critically after enrichment and expansion from human bone marrow: consequences for microfluidic cell sorting.

Miguel Xavier,Hywel Morgan,Daniel Spencer,María C De Andrés,Richard O C Oreffo

doi:10.1098/rsif.2017.0233

Abstract

The capacity of bone and cartilage to regenerate can be attributed to skeletal stem cells (SSCs) that reside within the bone marrow (BM). Given SSCs are rare and lack specific surface markers, antibody-based sorting has failed to deliver the cell purity required for clinical translation. Microfluidics offers new methods of isolating cells based on biophysical features including, but not limited to, size, electrical properties and stiffness. Here we report the characterization of the dielectric properties of unexpanded SSCs using single-cell microfluidic impedance cytometry (MIC). Unexpanded SSCs had a mean size of 9.0 µm; larger than the majority of BM cells. During expansion, often used to purify and increase the number of SSCs, cell size and membrane capacitance increased significantly, highlighting the importance of characterizing unaltered SSCs. In addition, MIC was used to track the osteogenic differentiation of SSCs and showed an increased membrane capacitance with differentiation. The electrical properties of primary SSCs were indistinct from other BM cells precluding its use as an isolation method. However, the current studies indicate that cell size in combination with another biophysical parameter, such as stiffness, could be used to design label-free devices for sorting SSCs with significant clinical impact.

Highlights

Skeletal stem cells (SSCs) are present in bone marrow (BM) and have the capacity to form fat, cartilage and bone
Unsorted human bone marrow mononuclear cells (hBMMNCs) were analysed by flow cytometry and a representative scatter plot (FSC-A versus SSC-A) of the sample is presented in figure 2a
If the size and membrane capacitance differences observed between expanded SSCs and other BM cells were verified for unexpanded SSCs, SSC isolation from human BM using microfluidic label-free sorting techniques could be achieved

Summary

Introduction

Skeletal stem cells (SSCs) are present in bone marrow (BM) and have the capacity to form fat, cartilage and bone Their differentiation capacity is restricted to skeletal lineages and the terms SSCs and mesenchymal stem cells (MSCs) have been used interchangeably, SSCs should be distinguished from MSCs which are reported to exist in extra-skeletal tissues and do not contribute to skeletal development [1,2,3]. At present there are no protocols for the isolation of a homogeneous SSC population from human BM [3] Despite their potential both in clinical applications [4,5] and for R&D, SSCs have only been used as a heterogeneous mixture of human bone marrow mononuclear cells (hBMMNCs) that include stem cells, other stromal progenitors and cells of the haematopoietic lineage.

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