Shape-dependent regulation of differentiation lineages of bone marrow-derived cells under cyclic stretch

Abstract

Multipotent stem cells are considered as a key material in regenerative medicine, and the understanding of the heterogeneity in the differentiation potentials of bone marrow-derived cells is important in the successful regenerative tissue repair. Therefore, the present study has been performed to investigate how the differentiation of post-harvest, native bone marrow-derived cells is regulated by cyclic stretch in vitro. Bone marrow-derived cells were obtained from mouse femur of both hind limbs and categorized into the following five categories: amebocytes, round cells, spindle cells, stellate cells and others. The cells were seeded on a silicone-made stretch chamber, and subjected to cyclic stretch with an amplitude of 10% at a frequency of 1 Hz for 7 days for cell shape analysis and for 3 days for the analysis of the expression of marker proteins of osteogenic (osteocalcin), vascular smooth muscle (α-smooth muscle actin and smooth muscle myosin heavy chain) and neurogenic (neurofilament) differentiation. When disregarding the differences in the cell shapes, there was an overall trend that the application of 10% cyclic stretch inhibited osteogenic and neurogenic differentiation, but enhanced smooth muscle differentiation. Close examinations revealed that round cells were influenced the most by cyclic stretch (significant up- or down-regulation in all the four marker protein expressions) while amebocytes and spindle cells were only influenced by cyclic stretch for vascular smooth muscle and/or neurogenic differentiation. As far as the authors know, this is the first study reporting the shape-related differences in the fate decision criteria for mechanical strain in bone marrow-derived cells.

Introduction

Stem cells have a multipotent differentiation capability and have been considered as a key material in the success of regenerative medicine. Bone marrow is a primary source of the stem cells among other tissues such as blood and adipose tissue. Bone marrow-derived stromal cells, also named as bone marrow mesenchymal stem cells (bMSCs), have been studied extensively for their multipotent differentiation capability. Moreover, an application of mechanical loading has been demonstrated to be effective in directing differentiation towards cells in load-bearing tissues such as blood vessel (vascular smooth muscle cells), bone (osteoblasts) and articular cartilage (articular chondrocytes) (Steward and Kelly, 2015).

Among a variety of mechanical loading regimens, cyclic stretch has been adopted in a number of studies in an attempt to lead bMSCs into specific lineages. It has been demonstrated that the application of cyclic stretch to bMSCs in vitro resulted in changes in the expression of markers for osteogenic, chondrogenic, smooth muscle and tenogenic differentiations. The direction of the change (i.e., upregulation or downregulation) depends on the amplitude of cyclic stretch. In general, cyclic stretch with a low amplitude up to 5% induces an upregulation of osteogenic differentiation (Byrne et al., 2008, Haudenschild et al., 2009, Kearney et al., 2010, Koike et al., 2005, Qi et al., 2008, Rui et al., 2011, Ward et al., 2007), whereas that with an amplitude over 10% resulted in the enhancement of the differentiation towards smooth muscle cells (Ghazanfari et al., 2009, Jang et al., 2011). Tenogenic differentiation is inducible by cyclic stretch with an amplitude ranging from 1% to 10% (Kuo and Tuan, 2008, Morita et al., 2019, Morita et al., 2013).

It is known that bone marrow contains cells with a variety of shapes. Two types of cell shape were reported first (Mets and Verdonk, 1981): fibroblast-like cells and large, epithelial-like cells. This was followed by a report showing the presence of round-shape cells (Colter et al., 2001, Kobayashi et al., 2004, Vogel et al., 2003). These cell shapes seemed to represent different differentiation capabilities; fibroblast-like spindle cells can be differentiated into smooth muscle cells under fluid flow stimulation, while round-shape cells and large cells can be differentiated into adipocytes and osteoblast, respectively (Kobayashi et al., 2004). Chondrogenic and neurogenic differentiation capabilities have also been confirmed (Freeman et al., 2015, Ward et al., 2007). Despite the presence of such heterogeneity in the population of bone marrow-derived cells, in the most of past studies, cells were isolated from the marrow of long bones, expanded and cultured in tissue culture flasks before the use in subsequent culture experiments for cell differentiation as stem cells (e.g., Both et al., 2007, Farrell et al., 2006). During these procedures, non-adherent cells, which could include hematopoietic cells, were excluded so that the remaining, cells attaching to a plastic substrate were selectively cultured to confluency, at least once, to make them a homogeneous population of stem cells. Although this could potentially give researchers stable and reproducible materials, such stem cells still demonstrate widely varying differentiation potentials between individual cells (Freeman et al., 2015). In addition, the use of stem cell lines comes with a drawback that the differentiation capability of the original, native cells in bone marrow cannot be analyzed. The heterogeneity of bone marrow-derived stem cells is thought to lead to an unsatisfactory outcome of tissue repair by the administration of these cell types in clinical settings (Huang et al., 2010, Wang et al., 2015). For a better outcome in the therapeutic use of stem cells, understanding of how the native bone marrow-derived cells can be differentiated to various specific lineages would be useful, particularly in the presence of extrinsic signals such as mechanical and/or chemical stimuli. Therefore, the present study has been performed to investigate how the differentiation of post-harvest, native bone marrow-derived cells is regulated by cyclic stretch in vitro.