AECM A PRESENTED BY: Malathi Srivatsan aECM Team Co-Lead 1 WHY - PowerPoint PPT Presentation

AECM A PRESENTED BY: Malathi Srivatsan aECM Team Co-Lead 1

WHY SURFACE ENGINEERING FOR CELL CULTURE? • 17,000 people sustain a spinal cord injury in the United States annually. • Neurons are killed or permanently damaged. • Unlike other cells, neurons do not divide to replace dead neurons. • To over come loss of function, Neural Progenitor Cells (NPCs) which have the ability to differentiate into neurons could be used for transplantation. Need improved methods for in vitro and in vivo application. TASK: Defining methods using surfaces to obtain enough functional new neurons to replace dead or damaged cells in nervous system A 2

Complexities in and around nervous system to be considered for promoting differentiation A 3

CASE is addressing the challenge by fabricating innovative growth surfaces for neuronal differentiation and cell to cell communication KEY ASSUMPTION : aECMs with tunable surfaces and added components will outperform simple platforms that currently exists for differentiating neural cells in vitro ( increased neuron number, viability, physiology). GOALS for Year II: • Year 2 Objective 1: Purchase, install and provide training on major equipment • Year 2 Objective 2: Develop aECM that promote differentiation into specific cell types • Year 2 Objective 3 Determine morphology of aECM and optimal structural interactions of tunable nanostructures with biological cells • Year 2 Objective 4: Fabricate and test 2D fiber and protein aECMs • Year 2 Objective 5: Develop contacts with Arkansas industry to promote commercialization of research • Year 2 Objective 6: Integrate research with education to increase next generation of scientists : A 4

Two major questions are being addressed: 2. Other sources (molecules, cells, organelles etc.) for 1. Neuronal Differentiation stem cell crosstalk/support of differentiation and from stem cells function Sequencing &Gene Reyna Griffin expression Srivatsan Borrelli Allen Biris Biris, Zou Ghosh Servoss APPROACH and TEAM: Using innovative surfaces to improve neuronal differentiation and viability/functionality: extracellular matrix and cell-cell A 5 communication are critical aspects

AECM TEAM CORE FACULTY A A Malathi Srivatsan Rob Griffin Team Co-Lead Team Co-Lead A A Shannon Servoss Nathan Reyna M Min Zou A Antiño Allen T Alexandru S. A Biris C Michael Borrelli Anindya Ghosh

CHANGES TO TEAM ACTIVITIES Experimental Approach: No major changes Personnel: Shiguang Yu (research faculty), replaced by postdoc at Arkansas State University. Robert J. Griffin (group leader), replaces Michael Borrelli as co-group leader with Dr. Srivatsan. A 7

MILESTONES AND OBJECTIVES-Yr II Objectives Milestones Status Purchase, install and provide training on Cell culture (UAMS) in yr 2 for aECM Milestone met major equipment Develop aECM that promote differentiation Quantify proportion of CNS ECM proteins Matrigel was identified to mimic natural CNS ECM. Some of its into specific cell types. that must be incorporated into aECMs properties were incorporated into first generation of aECM. Gold nanorod first generation surfaces tested. Optimizing multi-component surfaces with varying topography and efforts are continuing Determine morphology of aECM and Morphological and structural Milestone being met and efforts are continuing optimal structural interactions of tunable interactions measured using microscopy nanostructures with biological cells; (confocal, EM) and image analysis. develop simulation models for further optimization Fabricate and test 2D fiber and protein Most productive structural compositions To obtain most productive surface (surface that results in aECMs identified maximal neuronal differentiation), incorporation/addition of peptoids, exosomes, topographies, various protein coupling efforts are underway. Omics analysis platform established for exosomes or cells. Evaluations of the effectiveness of topographies, peptoids and exosomes are underway. Working groups for each curriculum kit will Curriculum kits planned and one of each develop the curriculum and supply lists for style being constructed. assigned kits Develop contacts with Arkansas industry to Industry visits to give insight on Developing collaboration with Carbon Nano onion, LLC promote commercialization of research successful startup in Arkansas and the iCorp program Integrate research with education to Create courses, incorporate research Met milestone and moving forward increase next generation of scientists related topics in existing courses 8 A

Obj.2 Develop aECM that promote differentiation into specific cell types- Control surfaces : Rat NPCs Differentiate well on Matrigel and laminin, two natural ECM Materials used as control surfaces: Srivatsan et al. Polylysine+Laminin (Low Magnification) Matrigel (High Magnification) Neuron = Beta 3 Tubulin Astroglia = GFAP Nuclei of all cells =DAPI Matrigel substratum promotes more neuronal differentiation compared to Poly-D-Lysine & Laminin 60 ** % Cell I differentiation 50 40 30 20 10 0 Astrocytes Neurons Different Surfaces Extracellular Matrix significantly increases neuronal differentiation A 9

Obj. 2 Develop aECM that promote differentiation into specific cell types- Control surface : Rat NPCs Differentiate well on Matrigel into Oligodendrocytes: Srivatsan et al. 120.0 ODC Astrocytes Undifferentiated 100.0 ** % of cells ** 80.0 60.0 40.0 20.0 0.0 PDL Matrigel Sequential addition of biomolecules along with the ECM surface significantly increase differentiation of ODCs at a faster rate.

Obj.2 Develop aECM that promote differentiation into specific cell 1 ST GEN SURFACE: Biris et al . types- Cartoon diagram of ~30 nm gold plasmonic nanorod double layer Electron Micrograph of Gold nanorod with incorporated carboxy (red dots) and amino (blue dots) groups surface A 11

Obj.3 Determine morphology of aECM and optimal structural interactions- Density and composition of gold matters : Rat Neural Stem Cells (18 Days) Grown on Gold Nanorods Coated with Laminin: Borrelli et al Blue: Nuclei Green: Neurons Red: Astrocytes First generation surface promising; stimulating design of second generation A with added components 12

Obj. 3 Determine aECM morphology, interactions with & effects on NSCs- Gold Nanorod surface and Neuronal Differentiation: Srivatsan & Biris et al. • Found to be not toxic to cells • Cells attach very well without any coating • Cells differentiate either into neurons or into astrocytes • Does not significantly increase neuronal differentiation • May need to be coupled to other materials to improve results AuNR surface is biocompatible, provides excellent adhesion and encourages differentiation of neurons as A well as astrocytes 13

Obj. 3 Determine aECM morphology, interactions with & effects on NSCs- Surfaces made from barium titanate crystals promoted growth and differentiation of NSCs without the need for any coating with laminin: Borrelli et al. • The real potential for the barium titanate surfaces is that they can be polarized readily in a constant or time-varying manner. Activating the barium titanate with time-varying waveforms will produce ultrasonic waves parallel (surface waves) or normal to the surface • Using barium titanate surfaces alone, or in combination with the other types of aECM surfaces offers the potential to use electrically- or pulsed laser-induced ultrasound into the aECM surfaces to stimulate NSC differentiation into neurons and 150 µM increase Neuronal plasticity Blue: Nuclei – Hoechst Dye Green: Neurons - Anti-Beta Tubulin III Red: Astrocytes – Anti-GFAP

Obj.4 Fabricate and test 2D fiber and protein aECMs Nano Cellulose surface for Neuronal Differentiation: Srivatsan & Ghosh et al. • Found to be not toxic Nano cellulose was to cells coupled to Lysine to provide + charged surface • Cells attach well • Cells differentiate mostly into neurons or • May be very suitable to be coupled to other materials to improve results A 15

Obj. 3 Determine aECM morphology, interactions with & effects on NSC differentiations- Comparative efficacy of the different surfaces tested for NSC differentiation: Srivatsan, Biris & Ghosh et al . Neuronal differentiation on AuNR surface was slightly higher compared to nanocellulose, however astrocyte differentiation was significantly lower on Nanocellulose Matrigel substratum promotes more neuronal differentiation compared to Poly-D-Lysine & Laminin 60 ** 50 * 60 * 40 50 % differentiation ** % differentiation 30 Matrigel 40 Poly-D-lysine + 30 20 Laminin AuNR Surface 20 10 Nanocellulose 10 0 Astrocytes Neurons 0 Different Surfaces Astrocytes Neurons Different Surfaces Matrigel, Nanocellulose and gold nanorod surfaces all promote neuronal differentiation between 44 to 50% of total progenitor cell population

Exosomes: 100 nm, thousands/day Exosomes are an important aspect of extracellular matrix and may influence neural differentiation. Our approach: use exosomes from varying cell types to affect positive change, and incorporate exosomes into growth matrix. Challenge: isolation of exosomes from primary cultures; initial work with serum-derived or transformed cell line exosomes. (Gupta and Pulliam Journal of Neuroinflammation 2014, 11:68) 17 A