High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in longterm in vivo transplant studies for nervous system rescue and repair

Patterning of wound-induced intercellular Ca2+ flashes in a developing epithelium

Differential mechanical force distributions are increasingly recognized to provide important feedback into the control of an organ's final size and shape. As a second messenger that integrates and relays mechanical information to the cell, calcium ions (Ca2+) are a prime candidate for providing important information on both the overall mechanical state of the tissue and resulting behavior at the individual-cell level during development. Still, how the spatiotemporal properties of Ca2+ transients reflect the underlying mechanical characteristics of tissues is still poorly understood. Here we use an established model system of an epithelial tissue, the Drosophila wing imaginal disc, to investigate how tissue properties impact the propagation of Ca2+ transients induced by laser ablation. The resulting intercellular Ca2+ flash is found to be mediated by inositol 1,4,5-trisphosphate and depends on gap junction communication. Further, we find that intercellular Ca2+ transients show spatially non-uniform characteristics across the proximal–distal axis of the larval wing imaginal disc, which exhibit a gradient in cell size and anisotropy. A computational model of Ca2+ transients is employed to identify the principle factors explaining the spatiotemporal patterning dynamics of intercellular Ca2+ flashes. The relative Ca2+ flash anisotropy is principally explained by local cell shape anisotropy. Further, Ca2+ velocities are relatively uniform throughout the wing disc, irrespective of cell size or anisotropy. This can be explained by the opposing effects of cell diameter and cell elongation on intercellular Ca2+ propagation. Thus, intercellular Ca2+ transients follow lines of mechanical tension at velocities that are largely independent of tissue heterogeneity and reflect the mechanical state of the underlying tissue

Capabilities and Limitations of Tissue Size Control through Passive Mechanical Forces

Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery; understanding this process requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. In this work, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the Drosophila embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been done to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by the tissue recoil after laser ablation experiments

Capabilities and limitations of tissue size control through passive mechanical forces

<div>Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells, as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery. Understanding such control mechanisms requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. </div><div><br></div><div>Here, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the <i>Drosophila</i> embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been carried out to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by tissue recoil after laser ablation.</div><div><br></div

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in longterm in vivo transplant studies for nervous system rescue and repair.This article is from Journal of Visualized Experiments (2015): e52242, doi: 10.3791/52242. Posted with permission.</p

Compartment size control can emerge from passive mechanical forces.

<p>(A) Snapshots of <i>wt</i>, <i>en>dap</i> and <i>en>CycE</i> simulations, each following the final round of division. Parameter values are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.t001" target="_blank">Table 1</a>. (B) Comparison of simulated P compartment areas and cell numbers with observed values [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.ref014" target="_blank">14</a>]. Mean values from 100 simulations are shown and error bars are standard deviations. (C) Variation of P compartment area (upper row) and cell number (middle row), and of the number of accumulated cell deaths in the <i>en>CycE</i> perturbation over 100 simulations in the front and back halves of the P compartment (lower row), as each mechanical parameter is varied individually, holding all other parameters at their values listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.t001" target="_blank">Table 1</a>. Shaded areas in (B) and (C) mark the ranges of experimentally observed values and are added for reference (see main text for details).</p

Cell area distributions in the <i>en>dap</i> and <i>en>CycE</i> perturbations are multimodal.

<p>Distributions of cell areas for each perturbation of cell division events (<i>wt</i>, <i>en>dap</i> and <i>en>CycE</i>) and each scenario of cellular asymmetry. Cell areas are recorded at the end of each simulation and error bars denote standard deviations across 100 simulations. Parameter values are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.t001" target="_blank">Table 1</a> and in the main text.</p

Spatial regulation of mechanical cell properties can induce asymmetry of cell death occurrence inside posterior compartments.

<p>(A) Schematic of the distinct forms of mechanical asymmetries considered in this work. (B) Snapshot of final configuration of simulations for each considered perturbation. (C) Comparison of P compartment areas and cell numbers for each of the considered perturbations with experimental values. Mean values from 100 simulations are shown and error bars are standard deviations. Parameter values are given in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.t001" target="_blank">Table 1</a> and in the main text. Shaded areas mark the ranges of experimentally observed values and are added for reference and comparison with <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.g003" target="_blank">Fig 3</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.s003" target="_blank">S1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.s004" target="_blank">S2</a> Figs. (D) Comparison of accumulated number cell deaths over 100 simulations in the front and back halves of the P compartment for each of the considered perturbations.</p

Sensitivity of P compartment size and cell number to asymmetry.

<p>Variation of P compartment area (upper row) and cell number (middle row), and of the number of accumulated cell deaths over 100 simulations in the front and back halves of the P compartment (lower row), as the asymmetry parameters λ_<i>A</i>, λ_<i>l</i>, and λ_<i>p</i> are varied individually while holding all other parameters at their values listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.t001" target="_blank">Table 1</a>. Shaded areas are added for comparison with Figs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.g003" target="_blank">3</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004679#pcbi.1004679.g005" target="_blank">5</a>.</p