An HA-Based Hydrogel System for Therapeutic Delivery of MSCs to the Contused Spinal Cord

1. An HA-Based Hydrogel System for Therapeutic Delivery of MSCs to the Contused Spinal Cord Erin Moffitt Project AIMS – PRELIM Preparation

2. Motivation • Each year approximately 12,000 Americans sustain spinal cord injury (SCI), with most injuries occurring between ages 16-30. Estimated lifetime cost (for severe injury at 25 yrs) is over $3 million (NSCISC, US annual report, 2008) • Approximately half (49%) of SCIs are classified as contusions or cavities; other classifications being general SCI (10%), laceration (21%), and massive compression (20%) (Bunge 1993, Norenburg 2004). • Existing methods of treatment • Spontaneous regeneration is extremely limited due to the local environment of the CNS (favoring fibrosis over regeneration). • Current research has investigated : • 1) Acute treatment involving pharmacological approaches to control ischemia, toxicity, inflammation using neuroprotective agents (Perale et al 2008, Horner et al 2000) • 2) Regenerative approaches involving injection of precursor cells, or scaffolds implanted post-glial scar removal to bridge the lesion (Perale et al 2008, McDonald et al 2002)

3. Proposal: Create a tunable HA-hydrogel for therapeutic delivery of MSCs to spinal cord contusion injury AIM I: Determine the influence of hydrogel elastic modulus (local and bulk) on neurotrophic factor secretion by MSCs • Measure local hydrogel elastic modulus via atomic force microscopy (AFM) • Measure bulk elastic modulus via rheology • Evaluate encapsulated cell response (in vitro) AIM II: Determine influence of biological signals on neurotrophic factor secretion by MSCs • Measure in vitro protein retention over time for elastic moduli characterized in AIM I • Evaluate encapsulated cell response (in vitro) AIM III: Evaluate regenerative effect of implanted gels on an injured rat spinal cord • Determine dura permeability to proteins/growth factors • Evaluate effects of GMHA gel implant on inflammation and angiogenesis • Evaluate effects of GMHA-MSCs on inflammation, angiogenesis, and functional recovery

4. Combinatorial Approach • Mesenchymal Stem Cells: MSCs are a promising cell type due to their accessibility, expandability, high plasticity and limited effects on the immune system, presenting the possibility of allogenic transplantation (Perale, 2008). They have been shown to release soluble factors that have anti-inflammatory effects (Oh et al, 2008) as well as variety of neuro-regulatory molecules, ECM proteins, and relevant growth factors (NGF, BDNF, VEGF) (Hardy, et al). • Hyaluronic acid: (HA), the basis for our scaffold design, is involved in cell proliferation, morphogenesis, inflammation, and wound repair. It supports MSC viability without the addition of ECM factors. We hope to see a synergistic effect on inflammation suppression by combining HA/MSCs. • Biodegradable 3D Hydrogel Scaffold: By controlling the scaffold composition and thus the cell microenvironment, we can direct the cells down a desired lineage. The 3-dimensionality of the scaffold allows for spatially controlled delivery of cells, with localized release of MSC-produced GFs.

5. AIM I: Determine the influence of hydrogel elastic moduli (local and bulk) on neurotrophic factor secretion by MSCs • Hypothesis: Hydrogel compositions with elastic moduli most similar to native neural tissue will upregulateneurotrophic factor secretion by MSCs • Rationale: Stem cell microenvironments are known to be crucial to their lineage specification. By modifying the mechanical properties of the gel, and thus the neurogenic transcripts, we hope to also vary the growth factor release profile of the MSCs to eventually control their effect on SCI. • Supporting studies: • Engler et al showed that MSCs growing on substrates similar in stiffness to native brain (0.1-1 kPa) show the greatest expression of neurogenic transcripts when compared to greater stiffnesses (10-100 kPa) (Engler et al, 2006) • Based on literature, we are most interested in looking at MSC production of NGF, BDNF, NT3 VEGF, and a variety of interleukins (Hardy et al, 2008)

6. AIM Ia: Measure local hydrogel elastic modulus via atomic force microscopy (AFM) • Experimental Plan: • Obtain force measurements for 3 gel compositions (N=3 per group) • Obtain force measurements for brain and spinal cord tissue of similar thickness (N=3 per group) • Derive corresponding elastic modulus for each sample • Preliminary Data: • GMHA gels of compositions tested this far show to be significantly different • Experiments will be repeated with three gel compositions and N=3 for each group

7. AIM Ib: Measure bulk elastic modulus via rheology • Experimental Plan: • Obtain storage moduli G′ and loss moduli G″ under constant temperature (37 °C) for 3 gel compositions corresponding to AIM Ia, (N=3 per group) • Compare elastic modulus determined in previous technique (AFM)

8. AIM II: Determine influence of biological signals on neurotrophic secretion by MSCs • Hypothesis: Altering concentration of ECM molecules (FN/LN), as well as growth media conditions, will change the cell micro-environment, therefore influencing MSC growth factor secretion • Rationale: Cellular interaction with its microenvironment leads to various degrees of attachment, spreading, and behavior. Modifying this environment by exposing cells to adhesive proteins or various growth medium may favorably alter cell viability, proliferation, or GF secretion. • Supporting studies: • Many groups have induced “transdifferentiation” of MSCs with the use of neural induction media (Woodbury 2000, Sanchez-Ramos 2000, Deng 2001, Qian 2004) • Chen et al showed that MSCs vary their secretion patterns depending on their microenvironment, and may amplify secretions in response to injury (Chen et al, 2002)

9. AIM IIa: Measure in vitro protein retention over time for elastic moduli characterized in AIM I • Experimental Plan: • Bioconjugate FITC to protein of interest (FN or LN) • Encapsulate known concentration of protein • Track protein leaching vs. retention at several time points • Preliminary Data: • Significantly more protein is retained in 11% (p<0.01) and 30%(p<0.01) gels than in the solutions • Study should be repeated with UV-treated groups for calibration curve (with more time points)

10. AIM Ic and IIb: Evaluate encapsulated cell response (in vitro) • Experimental Plan: • Evaluate encapsulated MSC viability (confocal + cell count) and proliferation (BrdU) in gels • RT-PCR to identify genes, indicative of GFs of interest (compared to cells on TCP) • Quantify neurotrophic secretion of identified GFs (ELISA) for various experimental groups • Preliminary Data: • Encapsulated MSCs are highly viable in 11% and 20% gels for at least 3-4 weeks (live/dead) • Cells begin spreading after 2 weeks, more widely seen in 11% gels control FN LN 11% 20%

11. AIM III: Evaluate regenerative effect of implanted gels with varying GF release profiles on an injured rat spinal cord • Hypothesis: Comparing several gel compositions with varied GF release profiles, we can isolate a desirable combination of factors for improved functional recovery of SCI. • Rationale: MSCs are known to provide functional benefit to SCI patients, though mechanisms for this improvement are unknown. One proposed mechanism is delayed or reduced inflammation (which may be enhanced by the addition of an HA-gel as seen in Zin’s data). Another is precise and temporal delivery of growth factors, a combination which is not well characterized. We hope to uncover some of these questions, while proposing a novel method for controlled delivery of MSCs • Supporting studies: MSCs improve functional outcome in cases of SCI (Hofstetter et al, 2001)

12. AIM IIIa: Evaluate permeability of SC dura to growth factors • Experimental Plan: • Obtain rat SC dura • Place in diffusivity chamber with proteins/GFs on one side of chamber • Use microBCA to evalulate protein content, ELISA for GF content • Outcome Measures: • Relative permeability of dura to biofactors of interest • If permeability is apparent, data should justify using a contusion model for delivery

13. AIM IIIb: Evaluate implantation of GMHA in contusion model • Experimental Plan: • Work with collaborators in Houston (Grill lab) and Zin to learn and utilize contusion model • Injure spinal cord (N=16 rats) • Rats receive GMHA gel implant to cover injury site, or no treatment • Spinal cords harvested at 2-3 weeks and sectioned for histological evaluation / immunohistochemistry • Outcome Measures: • Inflammation • T-cells (OX19, W3/25, OX8) • Macrophage infiltration (OX42, ED1) • Astrocyte infiltration (GFAP) • Angiogenesis • Endothelial cell staining (RECA1, Von Willebrand factor)

14. AIM IIIc: Evaluate implantation of GMHA-MSCs in contusion • Experimental Procedure: • Repeat contusion injury • Implant GMHA gel of varying composition (to be determined in AIMs I/II) • Monitor and evaluate rats for functional recovery using BBB and pain response • Harvest spinal cords at various time points (acute and longer) and section for histology • Compare to controls in AIM IIIa • Outcome Measures: • Inflammation • T-cells (OX19, W3/25, OX8) • Macrophage infiltration (OX42, ED1) • Astrocyte infiltration (GFAP) • Angiogenesis • Endothelial cell staining (RECA1, Von Willebrand factor) • Functional Recovery • BBB locomotor rating scale • Pain response (thermal sensory testing, or von Frey)

15. Proposal: Create a tunable HA-hydrogel for therapeutic delivery of MSCs to spinal cord contusion injury AIM I: Determine the influence of hydrogel elastic modulus (local and bulk) on neurotrophic factor secretion by MSCs • Measure local hydrogel elastic modulus via atomic force microscopy (AFM) • Measure bulk elastic modulus via rheology • Evaluate encapsulated cell response (in vitro) (in part) AIM II: Determine influence of biological signals on neurotrophic factor secretion by MSCs • Measure in vitro protein retention over time for elastic moduli characterized in AIM I • Evaluate encapsulated cell response (in vitro) (in part) AIM III: Evaluate regenerative effect of implanted gels on an injured rat spinal cord • Determine dura permeability to proteins/growth factors • Evaluate effects of GMHA gel implant on inflammation and angiogenesis • Evaluate effects of GMHA-MSCs on inflammation, angiogenesis, and functional recovery * Finish by June?

16. 2009 Project Timeline: Countdown to Prelims JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC AFM (5,10,20% gels) and SCI/brain tissue (Jon) Gel Rheometry (Shalu) LT Protein Release Study (Ana) MSC EncapsViability/ ProliferationAssays (Ana) Contusion surgeries/rat care (Zin) Spinal cord analysis (Zin) Literature Review Writing Submit to Committee Prelims

17. AIM I/II contingency: Vary growth media for potential MSC-neuronal induction • Experimental Plan: • Treat MSCs with purchased (Lonza) hMSC growth media vs. neuronal induction media • Compare to MSCs on TCPS for controls • Stain for early neural markers (nestin) and mature neuronal markers (MAP2, Tuj1), and glial markers (GFAP) • MSC Growth Medium (Lonza-Proprietary): Basal medium + “MCGS” (serum) + gentamicin + L-glutamine • Neural Induction Medium (Yim et al, Exp Cell Res 2007):MSC Growth Medium + retinoic acid