Space-Associated Stem and Hematopoietic Cell Aging in Astronauts on Short-Duration Missions
Authors: Pham Jessica , Balaian Larissa , Chang Patrick , Nandi Shuvro P , Sneifer Jenna S , Ruiz Antonio W , Klacking Emma E , Van Der Werf Inge , Mack Karla , Katragadda Neha , Engstrom Claire , Quintero Cristian , Isquith Jane , Mesci Pinar , Molina Elsa , Stoudemire Jana , Whisenant Thomas , Alexandrov Ludmil , Jamieson Catriona 
Published: December 2024

Introduction
The International Space Station (ISS) is a uniquely accelerating environment to study micro- and macro-environmental stressors in the context of modeling human diseases by simulating inflammation, aging, immune dysfunction, and accumulation of mutations. Studies done in low earth orbit (LEO) may offer accelerated insights into human health, and specifically, human hematopoietic stem cell (HSC) and immune cell health and function. Stem cells are resilient to environmental stressors: they can self-renew without differentiating, can differentiate into tissue-specific progenitors, and maintain longevity while retaining dormancy. As a follow-on study to ex-vivo nanobioreactor cultures sent to the ISS in four iterative 30-45 day missions, we hope to study the effects of spaceflight on the astronauts conducting these experiments in low earth orbit (LEO).
Methods
Under a NASA IRB-approved protocol, we collected peripheral blood from seven astronauts, who flew in Axiom Mission 2 and Axiom Mission 3 (Ax-2, Ax-3). CD34+ hematopoietic stem and progenitor cells (HSPCs) were isolated to identify functional, genomic, and single-cell transcriptomic changes across ten different timepoints by clonogenic assay, 90X whole genome sequencing (WGS), and single-cell RNA sequencing (scRNA-seq) respectively. To date, timepoints include two pre-flight, one during mission, and 2 (Ax-3) to 3 (Ax-2) post-flight. Parallel studies were also conducted to profile the immune subsets by fluorescence-activated cell sorting and scRNA-seq analysis.
Results
To investigate the effects of spaceflight on HSC function, we performed clonogenic survival and self-renewal assays. The percent of multilineage colonies from Ax-2 and Ax-3 was significantly reduced inflight compared to pre- and post-mission. HSPCs had significantly increased self-renewal capacity upon return (R + 1 day) in Ax-2. The self-renewal capacity of HSPCs from Ax-3 were significantly decreased inflight when compared to other timepoints. Interestingly, Ax-3 HSPCs begin to lose their self-renewal capacity in anticipation of launch at launch - 2 days (L - 2 days). Upon return, HSPCs appear to regain their cloning capacity after 30-days (R + 30 days). To corroborate these findings, scRNA-seq analyses revealed that while spaceflight induces significant changes in self-renewal genes, results indicate that a fight-or-flight response may be occurring immediately pre-mission. This suggests that changes seen inflight may be compounded by changes leading up to flight. Analysis is ongoing to identify mutational burden and mutational signatures associated with each timepoint, as well as targeted FACS analysis to profile the immune subsets.
Conclusion
Preliminary results from Ax-2 and Ax-3 show dynamic changes in stem cell function associated with pre-, during, and post-flight. Some changes to HSCs are individual-specific, indicating variable responses to environmental stressors. Functional and scRNA-seq analyses also suggest that duration spent in LEO is a contributing factor to HSC recovery. This longitudinal study has the potential to provide valuable insight into human health on Earth and inform on the effects of longer duration missions and future missions beyond the ISS.