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Material viscoelastic properties modulate the mesenchymal stem cell secretome for applications in hematopoietic recovery Frances D. Liu, Novalia Pishesha, Zhiyong Poon, Tanwi Kaushik, and Krystyn J Van Vliet ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00644 • Publication Date (Web): 08 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017
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ACS Biomaterials Science & Engineering
Material viscoelastic properties modulate the mesenchymal stem cell secretome for applications in hematopoietic recovery
Frances D. Liu,1,2 Novalia Pishesha,1,3 Zhiyong Poon,2 Tanwi Kaushik,2 Krystyn J. Van Vliet1,2,4*
1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
2
BioSystems and Micromechanics (BioSyM) Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, CREATE, Singapore 138602 3
4
Whitehead Institute for Biomedical Research, Cambridge, MA 02139
Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
Corresponding Author *E-mail:
[email protected]. Telephone: (617) 253-3315
Abstract
Human mesenchymal stem cells (MSCs) exhibit morphological and phenotypic changes that correlate with mechanical cues presented by the substratum material to which those cells adhere. Such mechanosensitivity has been explored in vitro to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair in vivo through paracrine signaling via secreted biomolecules. Here, we leveraged cell-material interactions in vitro to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation.
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Specifically, we varied the viscoelastic properties of cell culture-compatible polydimethylsiloxane (PDMS) substrata to demonstrate modulated MSC expression of biomolecules including osteopontin, a secreted phosphoprotein implicated in tissue repair and regeneration. We observed an approximate three-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli < 1 kPa) and highest ratio of loss to storage moduli (tan > 1). A specific subpopulation of these cells, shown previously to express increased osteopontin in vitro and to promote bone marrow recovery in vivo, also exhibited up to a five-fold increase in osteopontin expression when grown on compliant PDMS relative to heterogeneous MSCs on polystyrene. Importantly, this mechanically modulated increase in protein expression preceded detectable changes in terminal differentiation capacity of MSCs. In co-culture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted in vitro proliferation of HSPCs without altering the multipotency for either myeloid or lymphoid lineages. Cytokine and protein expression by human MSCs can thus be manipulated directly by mechanical cues conferred by the material substrata, prior to and instead of tissue lineage differentiation. This approach enables enhanced in vitro production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications.
Keywords: human mesenchymal stem cells; secretome; cell-material interactions; osteopontin; hematopoiesis; bone marrow regeneration
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1. Introduction
Mesenchymal stem cells (MSCs) are a non-hematopoietic stem cell that can be obtained as a subset of bone marrow stromal cells.1 As MSCs can be induced in vitro to differentiate along osteogenic, adipogenic, and chondrogenic lineages,2 these cells have long been considered for in vitro organoids or tissue-engineered constructs and for delivery for direct tissue repair.3,4 These direct repair mechanisms would proceed in vivo presumably via MSC homing, engraftment, and differentiation into cell types required of the damaged tissue.5–8 Indeed, most in vitro studies have focused on mechanical modulation of phenotypic lineage commitment, e.g., via populationlevel expression correlated with differentiation along at least one mesenchymal tissue cell lineage, as a function of elastic modulus or stiffness of the materials to which the MSCs adhered.9–14 However, in vivo studies of systemically administered MSCs have demonstrated repair following local injury due to thrombotic stroke,15,16 myocardial infarction,17–19 and bone marrow irradiation.20 However, tissue repair can occur in some contexts even when MSC engraftment and differentiation are not detectable,20 and evidence for robust MSC differentiation at the injury sites remains a point of debate.20–24 Such studies suggest that MSCs can play an important indirect repair role via paracrine signaling, through secretion of immunomodulatory and pro-angiogenic cytokines to recruit and promote other cell types to repair the stroma of the injured tissue.25–33 Thus, there is growing interest in characterizing and manufacturing MSCs – in contrast to the progenitor or lineage-committed cells derived from MSCs – as a vehicle for indirect repair of bone marrow, neurological disorders, cardiovascular disease, liver failure, and immune disorders.24,25,29,34–40
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In our own work, we have exploited the indirect repair mechanisms of MSCs to support hematopoietic recovery in vivo.20 MSCs constitute a heterogeneous population of cells, at least upon in vitro expansion conditions employed typically for bench-scale research or clinical administration of adult human bone marrow-derived MSCs. This emergent population heterogeneity results in multiple subpopulations of mesenchymal stromal cells that differ in biophysical, in vitro, and in vivo properties,41–43despite undetectable changes in proposed immunophenotypic markers of stemness by the International Society for Cellular Therapy.44 We leveraged microfluidic sorting to enrich cell diameter-defined subpopulations of these mesenchymal stromal cells,41,45 effectively separating human MSCs (Dlo cells) from osteochondral progenitors of more restricted differentiation potential. This osteochondral progenitor subpopulation, defined in part by its relatively larger cell diameter (Dhi cells),41 homed to and promoted in vivo repair of the bone marrow compartment post-irradiation without sustained engraftment.20 That subpopulation secreted increased concentrations of growth factors and cytokines (e.g., ANG-1, BMP2, IL-8, and VEGF-A) known to promote hematopoietic recovery, and was consistent with prior reports of osteoblast-like cells or osteoprogenitors priming and organizing the hematopoietic microenvironment for hematopoiesis.46–49 Moreover, MSCs are not known to differentiate into or repopulate the hematopoietic cell lineages of the bone marrow, generally attributed instead to proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). Those findings support the concept that putative MSCs – more accurately described as multipotent mesenchymal stromal cells or as MSC-derived progenitors – can promote bone marrow repair indirectly by acting as “cellular factories” that produce secreted factors promoting HSPC growth and differentiation (see SI for further discussion).
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Obtaining sufficient numbers of these microfludically isolated MSC-derived osteochondral progenitors, or Dhi cells of larger diameter ~20 m, is inefficient because these cells constitute
