Icon Legend

This session is not in your schedule.

This session is in your schedule. Click again to remove it.

Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster V9 - Organ Preservation Using iPSC-MSC-Derived Extracellular Vesicles Toward Enhanced Transplantation Outcomes

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Bingda Li (he/him/his)

PhD Candidate in Biomedical Engineering
Carnegie Mellon University
Pittsburgh, Pennsylvania, United States

Primary Investigator(s)

PC

Phil Campbell

Research Professor in the Department of Biomedical Engineering
Carnegie Mellon University, United States

Co-Author(s)

EO

Ellen Otto

Introduction: Marginal organs from Extended Criteria Donors (ECDs) are less than optimal for addressing transplant organ shortages due to their heightened susceptibility to ischemia-reperfusion injury and immunological rejection. Extracellular vesicles (EVs) derived from iPSC-mesenchymal stem cells (iPSC-MSCs) exhibit promising capabilities in tissue regeneration, antioxidant activity, and immunoregulation, and have the potential to improve transplant outcomes with marginal organs. However, the mechanistic insights of EVs for organ preservation and the influence of temperature on EV therapeutics remain unexplored. This study investigates EV trafficking in tissue and explores the potential for organ reconditioning by supplementing the preservation solution with iPSC-MSC-EVs. Precision-cut lung slices (PCLS) are used as a high-throughput 3D model, closely mimicking the organ's in vivo environment, enabling efficient testing of diverse preservation strategies and analysis of EV uptake. EV trafficking within the tissue is analyzed through fluorescence microscopy and quantification of ¹²⁵I radiolabeled EVs. To unravel the therapeutic impacts of iPSC-MSC-EVs on tissue preservation, PCLS are treated with iPSC-MSC-EVs under hypoxic conditions to induce ischemic injury and inflammatory conditions induced by LPS. Preliminary findings from the uptake assay demonstrate the internalization of EVs into the tissue and the effect of temperature on EV internalization. Additionally, viability and inflammation assays indicate that iPSC-MSC-EVs exhibit cellular protection against ischemic conditions and immunoregulatory effects. Therefore, supplementing the preservation solution with iPSC-MSC-EVs for organ reconditioning may mitigate ischemic injury and immunological responses, improving transplantation outcomes.

Materials and

Methods: Precision Cut Lung Slice Collection:
C57BL/6 mice aged 8-15 weeks were euthanized using CO₂ asphyxiation, and the lungs were subsequently harvested. To maintain inflation, the lungs were infused with low melting point agarose (LMPA). Each lobe was excised and stored in ice-cold PBS. Lung lobes were sectioned into smaller blocks and precision-cut into slices using a Leica VT1000 vibratome. PCLS viability was assessed prior to experimentation.

iPSC-MSC-EV Functional Assay:
EV uptake assays were performed by incubating fluorescently labeled EVs with MRC-5 cells, followed by fluorescent microscopy to visualize EV internalization. For the EV inflammation assays, EVs were incubated with Rawblue cells, which were then exposed to LPS to induce an inflammatory response. The anti-inflammatory effects of the EV-pretreated cells were subsequently assessed.

EV Trafficking Analysis:
EVs were radiolabeled with ¹²⁵I, and trafficking analysis was conducted using PCLS. The trafficking of radiolabeled EVs was quantified using a gamma counter. ¹²⁵I-radiolabeled EVs were incubated with PCLS for 1 hour, after which surface-bound and internalized EVs were quantified. Additionally, EVs were fluorescently labeled with DiD and incubated with PCLS for 6 hours. Fluorescent images were taken to visualize EV uptake in the tissue.

iPSC-MSC-EV for Ischemic Injury:
Cobalt chloride (CoCl₂) was utilized as a hypoxia mimic to induce ischemic injury in PCLS. EV treatments were applied, and changes in metabolic activity were measured using a resazurin-based assay.

iPSC-MSC-EV for Immunoregulation:
LPS was employed to induce an inflammatory response in PCLS. EV treatments were administered, and the resulting inflammatory response was quantified using ELISA.

Results, Conclusions, and Discussions: Results

PCLS Viability:
PCLS were successfully obtained and remained viable for over 72 hours. The PCLS had a thickness of approximately 300 µm. Viability was assessed using the AlamarBlue assay, LDH assay, and live/dead (L/D) assay. The viability of PCLS stabilized after 24 hours and remained high for up to 72 hours.

Uptake and Anti-inflammatory Efficacy of iPSC-MSC-EVs:
iPSC-MSC-EVs, labeled with DiD fluorescent dye, were successfully internalized by MRC-5 cells, accumulating around the nucleus. Inflammation assays with Rawblue cells demonstrated that pretreatment with iPSC-MSC-EVs prior to LPS-induced inflammation significantly reduced the inflammatory response.

iPSC-MSC-EV Uptake in PCLS:
Based on fluorescent images, the EVs were successfully taken up by the PCLS. The ¹²⁵I-EV study indicated that PCLS cultured at 4°C and 10°C exhibited enhanced EV internalization compared to higher temperatures.

Protection Against Ischemic Injury:
LDH assays showed that treating PCLS with iPSC-MSC-EVs during hypoxia reduced cell death. Moreover, cold temperatures (4°C) provided better protection against ischemic injury than physiological temperature (37°C).

Reduction of Inflammatory Response:
PCLS treated with iPSC-MSC-EVs showed a reduced inflammatory response to LPS-induced inflammation, as measured by TNF-alpha quantification using ELISA.

Conclusion and Discussion
Overall, the preliminary results of this study demonstrate that iPSC-MSC-EVs can be successfully internalized by cells, especially under cold conditions, which is clinically relevant. Additionally, iPSC-MSC-EVs showed therapeutic benefits against ischemic injury in PCLS under hypoxic conditions induced by CoCl₂ and exhibited anti-inflammatory efficacy under LPS-induced inflammatory conditions.
Future steps include perfusing the whole lung with an EV-supplemented preservation solution, followed by comprehensive uptake and therapeutic analyses to further elucidate the mechanistic and therapeutic insights of EV therapy for organ preservation, with the aim of improving transplantation outcomes.

Acknowledgements (Optional):