The Ex-Vivo Human Translaminar Autonomous System to Study SANS Pathogenesis

Focused Investigation Project

During space travel, astronauts have to deal with long term exposure to zero gravity, ionizing cosmic rays, hypoxia, etc. The adverse effects of these challenging conditions result in a baffling physiological disorder such as Spaceflight Associated Neuro-ocular Syndrome (SANS). To understand the progression of SANS, in vitro human cells-based 3D biomimetic models capable of mimicking some of these adverse ocular symptoms is highly desired. This project aims to: 1) Differentiate retinal ganglion cells from human commercially-cleared induced pluripotent stem cells (iPSCs) based on published protocols and characterize phenotypes; 2) Adapt optimal differentiation protocols for spheroid neuro-glial co-culture based on experience with other human cell types and; 3) Establish a 3D co-culture system that mimics optic nerve tissue by directing axonal outgrowth within a micropatterned hydrogel.

There is an unmet clinical need for a new preclinical human model that can target disease etiology ex-vivo using intraocular pressure (IOP)/intracranial (ICP) to mimic the in-vivo paradigm of Spaceflight Associated Neuro-ocular Syndrome (SANS). We have successfully developed a two-chamber Translaminar Autonomous System (TAS) (U.S. patent application number 16/395610) that can independently regulate IOP and ICP using human donor posterior eyes. We can maintain tissue viability, integrity, complexity, and retinal functionality within the ex-vivo TAS model. The Translational Research Institute for Space Health (TRISH) funding allowed our research to have a high impact through a variety of modalities:

A) Impact on development of research model. Modifications performed on the TAS model to increase accuracy, efficiency, and productivity of research data.

1. We generated a new model design that allows a 16-chamber system and capacity to analyze 8 donor eyes within a single experimental run.

2. We built individualized pumps for each ICP and IOP chamber to modulate pressures with precision.

3. We utilized Arduino boards for automation of perfusion pumps to increase accuracy of pressure maintenance.

B) Impact on new prototype technologies: Development of the TAS Metrics software to utilize the ARKit platform to enable virtual measure using a mobile device's camera feature. High resolution photographic images can be captured of each posterior globe before and after each experimental run. The analyzed measures will include width and depth of the globe.

C) Scholarly Impact.

Patents: Utility patent application filed on April 26, 2019 under U.S. Patent Application No. 16/395,610. (already filed before TRISH funding but impacted further development) https://patents.google.com/patent/US20190327958A1/en?oq=16%2f395%2c610Placeholder

Publications: In preparation for submission and data collected from TRISH funds

1. The Ex-Vivo Human Translaminar Autonomous System to Study SANS Pathogenesis. Stacy M. Curry, Husain Lohawala, Gaurav Sharma, Tasneem P. Sharma.

2. Large scale application of the Ex-Vivo Human Translaminar Autonomous System. Husain Lohawala, Gaurav Sharma, Tasneem P. Sharma

D) Media and TRISH awareness impact.

1. https://www.unthsc.edu/newsroom/story/researcher-studies-the-impact-of-pace-on-the-human-eye/

2. https://dallasinnovates.com/astronauts-and-startups-how-a-unthsc-vision-scientists-device-could-help-how-we-see/

E) Impact on prospective future collaborations.

Potential collaborations made at NASA's Human Research Program (HRP) annual Investigators’ Workshop (IWS) and through various TRISH meetings:

1. Dr. Afshin Beheshti - miRNA project - NSPIRES program grant

2. Dr. Xiao wen Mao- radiation- Space Biology, HRP Impact funding mechanisms: Ability to apply for solicitations to NASA or other consortium based Human research project grants (BRASH 2101, NSPIRES awards) due to preliminary data generated from TRISH funds. We propose: Proposal 1: We will utilize the TAS model to study various simulated SANS pathogenic microenvironments using induced pluripotent stem cells: i. Aim 1) To determine effects of chronic mildly elevated ICP. ii. Aim 2) To determine effects of environmental factors. iii. Aim 3) To identify effective countermeasure strategies. Proposal 2: We will use conditioned medium from the TAS model and rodent biospecimens (undergoing basal, ground control, and flight-terminated research) to identify and compare pathogenic biomarkers from various simulated SANS pathogenic microenvironments: i. Aim 1) To characterize biomarkers within the IOP and ICP chamber conditioned medium of the TAS model. ii. Aim 2) To characterize biomarkers within spines and cerebral spinal fluid of rodents. iii. Aim 3) To contrast biomarkers from IOP and ICP chamber conditioned medium of the TAS model to spines and cerebral spinal fluid of rodents

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