Cytoskeletal Changes of Mesenchymal Stem Cells During Differentiation

Gregory Yourek

Mohammad Hussain

Jeremy Mao

^{Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois}

^{Department of Biotechnology, P.A. College of Engineering, Mangalore, India}

^{College of Dental Medicine, Columbia University, New York, New York}

Reprint requests: Jeremy J. Mao, DDS, PhD, Columbia University Medical Center, 630 West 168 Street - PH7 East - CDM, New York, NY 10032

Abstract

Mesenchymal stem cells (MSCs) are progenitors for tissues such as bone and cartilage. In this report, the actin cytoskeleton and nanomechanobiology of human mesenchymal stem cells (hMSCs) were studied using fluorescence microscopy and atomic force microscopy (AFM). Human MSCs were differentiated into chondrocytes and osteoblasts as per previous approaches. Cytochalasin D (CytD) was used to temporarily disrupt cytoskeleton in hMSCs, hMSC-chondrocytes (hMSC-Cys) and hMSC-osteoblasts (hMSC-Obs). Fluorescence microscopy revealed a dose-dependent response to CytD. Removal of CytD from the media of cytoskeleton-disrupted cells led to the recovery of the cytoskeletal structures, as confirmed by both fluorescence microscopy and AFM. Force-volume imaging by AFM evaluated the nanomechanics of all three cell types before, during, and after CytD treatment. Cytochalasin D disruption of cytoskeleton had marked effects on hMSCs and hMSC-Cys, in comparison with limited cytoskeleton disruption in hMSC-Obs, as confirmed qualitatively by fluorescence microscopy and quantitatively by AFM. Treatment with CytD resulted in morphology changes of all cell types, with significant decreases in the observed Young’s Moduli of hMSCs and hMSC-Cys. These data suggest human mesenchymal stem cells alter their cytoskeletal components during differentiation. Additional studies will address the mechanisms of cytoskeletal changes using biochemical and biophysical methods.

Abstract

Mesenchymal stem cells (MSCs) are self-renewing and multipotential cells with the capacity to differentiate into several distinct end-stage cell lineages that form bone, cartilage, adipose, tendon, muscle, neural, and other connective tissues.¹6 There is much interest in the potential of MSCs in tissue engineering of bone and cartilage for the treatment of musculoskeletal trauma and disease. Adult MSCs offer certain advantages over embryonic stem cells, including their readiness and availability, because they can be obtained from the same individual.⁷ Since their first description,⁸ MSCs have been shown to possess remarkable capacity for self-replication⁴ and multilineage differentiation capacity. They can be differentiated into bone- and cartilage-forming cells in the presence of chemical supplements and/or bioactive factors.³9 Potential applications of MSCs towards regeneration and treatment have been reported, such as for tissue-engineered mandibular condyle,¹⁰ total jaw,¹¹ osteogenesis imperfecta,¹² cardiac regeneration, ¹³ metachromatic leukodystrophy, and Hurler syndrome. ¹⁴ It has been proposed that the cytoskeleton may play a role in the differentiation of MSCs.¹⁵

The cytoskeleton plays important roles in cell morphology, adhesion, growth, and signaling. Changes in the cytoskeleton of the cell allow the cell to migrate, divide, and maintain its shape,¹⁶ and the cytoskeleton responds to external mechanical stimuli.¹⁷ The cytoskeleton consists of three components: actin filaments, intermediate filaments, and microtubules. The backbone of the cytoskeleton is F-actin, which clusters to form actin filaments. Filaments can be bundled and crosslinked by several actin-binding proteins in a network and are most likely anchored to stable structures, or anchor sites, in the cell (such as the plasma membrane).¹⁸ The actin network plays a major role in the determination of the mechanical properties of living cells¹⁹20 by forming a direct link between the integrins and the nucleus, which mechanically stiffens the nucleus and holds it in place.²¹

The atomic force microscope (AFM) was developed in 1986 by modifying the scanning tunneling microscope.²² Since the early use of AFM for imaging living cells, the subsurface cytoskeletal structures have been observed and described in the nanometer-scale range.²³24 The portion of the cytoskeleton most readily resolved by the AFM is actin filaments.²⁵ The conjunction of the AFM with other imaging techniques has also confirmed the ability to study microtubules and intermediate filaments with the AFM.²⁶27 It has been demonstrated that tightly adherent cells are more stiff than cells that are loosely attached,²⁸ suggesting a dynamic reorganization of the cytoskeletal elements is induced by the cellular attachment to the substrate. Upon study of the three cytoskeletal elements with immunofluorescent dyes using confocal laser scanning microscopy, the elasticity of the cell membrane was found to be related to the distribution of the actin and intermediate filaments, but much less to the microtubules.²⁶ Similar observations in two fibroblast cell lines confirmed the crucial importance of the actin filament network for the mechanical stability of living cells.¹⁹ The disruption of actin filaments causes a decrease in the average elastic modulus of the cell membrane, induced disassembly of microtubules has little effect on cell membrane elasticity.¹⁹

Although the biochemical events that occur upon stem cell differentiation are well-characterized, there is limited knowledge of the nanocharacteristics of undifferentiated and differentiating human mesenchymal stem cells (hMSCs). We have recently characterized the Young’s Modulus and surface roughness of differentiating hMSCs²⁹; however, how these characteristics relate to the underlying actin cytoskeleton of these cells is unknown. The physical characteristics of a cell have proven to be valuable features of cells; different types of cells have evident variations. We have chosen to focus on Young’s Modulus because of the significant differences between undifferentiating and differentiating MSCs found in our laboratory.²⁹

The hypothesis of this study was that structural changes associated with human MSC differentiation are actin-cytoskeleton dependent. There are numerous reports of gene expression and protein modifications of hMSCs, and of changes in morphology as differentiation occurs; however, little is known about how the structural aspects of these cells are modified as a result of differentiation. We investigated changes in cytoskeletal and nanomechanical characteristics of hMSCs after differentiation using fluorescence and atomic force microscopy techniques, respectively, in combination with cytoskeletal perturbation.

Footnotes

Presented in part at the 52nd Annual ASAIO Conference, June 8–10, 2006, in Chicago, Illinois.

Footnotes

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