MECHANICAL AND BIOMOLECULAR CHARACTERIZATION OF HUMAN BONE MARROW

Master'sMASTER OF SCIENC

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

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.National University of Singapore (Graduate School for Integrative Sciences and Engineering Program)Singapore-MIT Alliance (Singapore-MIT Alliance-3 graduate fellowship program)Singapore. National Research FoundationSingapore-MIT Alliance for Research and Technology (BioSystems and Micromechanics Interdisciplinary Research Group)Singapore. National Medical Research Council (NMRC/Clinician Scientist Award/012/2009

Material Viscoelastic Properties Modulate the Mesenchymal Stem Cell Secretome for Applications in Hematopoietic Recovery

© 2017 American Chemical Society. 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. 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 approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli 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 5-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 the terminal differentiation capacity of MSCs. In coculture 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

Novel Strategy for Selection of Monoclonal Antibodies Against Highly Conserved Antigens: Phage Library Panning Against Ephrin-B2 Displayed on Yeast

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as ‘EC8’) was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeuti

Material Viscoelastic Properties Modulate the Mesenchymal Stem Cell Secretome for Applications in Hematopoietic Recovery

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 <i>in vitro</i> to promote differentiation of MSCs along tissue cell lineages for direct tissue repair. However, MSCs are increasingly understood to facilitate indirect tissue repair <i>in vivo</i> through paracrine signaling via secreted biomolecules. Here we leveraged cell–material interactions <i>in vitro</i> to induce human bone marrow-derived MSCs to preferentially secrete factors that are beneficial to hematopoietic cell proliferation. 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 approximately 3-fold increase in expression of osteopontin for MSCs on PDMS substrata of lowest stiffness (elastic moduli <1 kPa) and highest ratio of loss modulus to storage modulus (tan­(δ) > 1). A specific subpopulation of these cells, shown previously to express increased osteopontin <i>in vitro</i> and to promote bone marrow recovery <i>in vivo</i>, also exhibited up to a 5-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 the terminal differentiation capacity of MSCs. In coculture with human CD34+ hematopoietic stem and progenitor cells (HSPCs) that repopulate the blood cell lineages, these mechanically modulated MSCs promoted <i>in vitro</i> 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 <i>in vitro</i> production of both mesenchymal and hematopoietic stem and progenitor cells that aid regenerative clinical applications

Detection of ephrin-B2 expression in human cancer cell lines and tumor xenograft in mice.

<p>(A) Flow cytometry measurements of EC8 binding to COLO205 and HCT116 cells (solid line) in comparison to the isotype control (shaded area). CHO cells with no ephrin-B2 expression were also included for comparison. (B) RT-PCR detection of ephrin-B2 expression in different cell lines. (C) Immunostaining of ephrin-B2 on human colon cancer xenografted in mice. Control denotes immunostaining without EC8 as a primary antibody. Tumor and stromal cells were indicated with arrowhead and arrow, respectively. Circle indicates murine endothelium stained with EC8. Scale bar = 20 µm.</p

Detection of ephrin-B2 expression in human tissue arrays.

<p>(A&B) Immunostaining of ephrin-B2 expression in human tumor tissue arrays using EC8. Control denotes immunostaining without EC8 as a primary antibody. Tumor and stromal cells were indicated with arrowhead and arrow, respectively. Scale bar = 20 µm. PAC = Papillary Adenocarcinoma; AC = Adenocarcinoma; BR = Bronchus; AL = Alveoli; SC = Squamous Cell Carcinoma; SAC = Serous Adenocarcinoma. IDC = Nonspecific Infiltrating Duct Carcinoma. (C) Immunofluorescence staining on human colon tumor tissue demonstrating that EC8 (red) detects ephrin-B2 expressions in both cancer cells and tumor-associated vasculature (green). Blow up views of the two areas indicated with dashed box are shown in the right panel. Scale bar = 100 µm.</p

Selection, validation, and sequence of ephrin-B2-specific human single-chain antibodies.

<p>(A) A schematic diagram of phage panning against antigens expressed in yeast display system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030680#pone.0030680-Boder1" target="_blank">[34]</a>. (B) Immunofluorescence flow cytometry measurements of protein and phage binding to yeast cells. Surface-displayed ephrin-B2 was detected by the binding of anti-Myc antibody (‘Myc’) as well as recombinant human EphB4-Fc (‘EphB4’) to yeast cells (top panel). Progressive enrichment of phage clones from first three rounds of panning (denoted as ‘1 st’, ‘2 nd’ and ‘3 rd’) was detected by antibody against His tag (bottom panel). Histograms drawn in shaded area and solid lines indicate antibody binding to uninduced and induced yeast cells, respectively. The percentage of phage clones with positive binding is indicated. (C) SDS-PAGE of scFv-EB1 (lane ‘1’) and scFv-EC8 (lane ‘2’). (D) Ephrin-B2 specific scFv binding to irrelevant yeast cells, yeast cells with expression of ephrin-B2 ectodomain, 293T cells, and 293T cells with transient expression of full-length ephrin-B2. Shown are the histograms of cells labeled with secondary antibody with (solid line) and without (shaded area) ephrin-B2 specific scFv as primary antibody. (E) Sequence alignment of scFv-EA6, scFv-EB1, and scFv-EC8. Complementarity determining regions (CDR), the beginning of immunoglobulin variable heavy (VH) and variable light (VL) chain domains, and the linker connecting VH and VL are noted. <b>*</b> indicates amino acids differ between scFvs.</p