title: Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency.
abstract: The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.
authors.affiliation: National University of Singapore Graduate School for Integrative Sciences and Engineering, Singapore 119077; BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602;
authors.forename: Wong Cheng
authors.initials: WC
authors.lastname: Lee
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602;
authors.forename: Hui
authors.initials: H
authors.lastname: Shi
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602;
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Poon
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602;
authors.forename: Lin Myint
authors.initials: LM
authors.lastname: Nyan
authors.affiliation: Department of Obstetrics and Gynaecology, National University of Singapore, Singapore 119228;
authors.forename: Tanwi
authors.initials: T
authors.lastname: Kaushik
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602; Department of Biological Sciences, National University of Singapore, Singapore 117543; Mechanobiology Institute, Singapore 117411;
authors.forename: G V
authors.initials: GV
authors.lastname: Shivashankar
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602; Department of Obstetrics and Gynaecology, National University of Singapore, Singapore 119228; Division of Reproductive Medicine, KK Women's and Children's Hospital, Singapore 229899; Duke-National University of Singapore, Singapore 169857;
authors.forename: Jerry K Y
authors.initials: JK
authors.lastname: Chan
authors.affiliation: National University of Singapore Graduate School for Integrative Sciences and Engineering, Singapore 119077; BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602; Mechanobiology Institute, Singapore 117411; Department of Biomedical Engineering, National University of Singapore, Singapore 117575;
authors.forename: Chwee Teck
authors.initials: CT
authors.lastname: Lim
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602; Departments of Electrical Engineering and Computer Science, Biological Engineering, and.
authors.forename: Jongyoon
authors.initials: J
authors.lastname: Han
authors.affiliation: BioSystems and Micromechanics Interdisciplinary Research Group, Singapore-MIT Alliance in Research and Technology, Singapore 138602; Biological Engineering, and Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 krystyn@mit.edu.
authors.forename: Krystyn J
authors.initials: KJ
authors.lastname: Van Vliet
mesh_terms: D000328:Adult; D000954:Antigens, Surface / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D055592:Biophysical Phenomena; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005333:Fetus / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D046210:Microfluidic Analytical Techniques; D039902:Multipotent Stem Cells / Q000166:cytology*; D015999:Multivariate Analysis; D009685:Nuclear Envelope / Q000378:metabolism; D013314:Stress, Mechanical
chemical_list: D000954:Antigens, Surface; D015415:Biomarkers
keywords: biomarkers; biophysical characterization; mesenchymal stem cell enrichment; multipotency; stem cell heterogeneity