This proposal is based on our robust preliminary data demonstrating that conditions mimicking microgravity (rotating wall vessel culture, RWVC) markedly alter human MK morphology and gene expression. We hypothesize that microgravity will re-program MKs and newly-released PLTs, resulting in critical changes in their transcriptome, proteome, and alterations in PLT number and function. We will determine how microgravity and space radiation conditions on board the International Space Station (ISS) alter human MK and PLT maturation/production, gene expression (DNA, RNA, and protein), and cellular function. We will study in vitro human hematopoietic progenitor cell (HPC)-derived MKs in Earth-based experiments under standard or microgravity conditions. In parallel, human MKs will be studied on the ISS. Integrated, cutting-edge OMICS toolsets (e.g., RNA-sequencing and ribosomal footprinting [Ribo-seq]), comprehensive morphologic studies, and cell production kinetic studies will be used. They will provide unprecedented insight into adaptation processes needed for MK and PLT function under conditions experienced by humans performing spaceflights. These studies will directly address crew health concerns that currently limit human space exploration and will assist in developing targeted countermeasures.

This proposal concurs with the major National Research Council (NRC) Decadal Survey Recommendations for cellular and molecular biology studies using state- of-the-art tools coupled with systems biology, and for studies evaluating the physiological interplay of cardiopulmonary and immune functions during application of spaceflight. Furthermore, we will address goals of the NASA Space Biology Science Plan 2016-2025, including: (1) determine the effects of the space environment on DNA function, (2) develop a systems biology-based understanding of the cellular and molecular changes to explain how gravitational changes in spaceflight effects organisms and causes phenotypic changes, and (3) identify how spaceflight affects the ability of cells to generate and maintain their complex internal cyto-architecture, processes critical for MKs and PLTs.

• Administrative/Organizational accomplishments: o The flight hardware solicitation was posted, flight hardware was selected and kick-off meeting happened took place on 07/13/2023. o The PI and Project Scientist prepared and finished the Science Requirement Document. o After the PI team was granted IRB exempt status, travel and experimental procedures to and at NSRL were administratively organized, and NSRL irradiation was performed. A rerun was granted by ASBCB, and is scheduled for Fall 2024. o NASA IRB approved our human subject research application needed for Specific Aim 3. In addition, the University of Utah IRB accepted the NASA IRB as single IRB of record, while NASA IRB accepted the University of Utah as a study site for aforementioned IRB. o NASA IRB was extended for an additional year. o Mini-ED and crew briefing materials were submitted and approved. o Two flight crews were briefed. o The PI, the Project Scientists, and the implementation partner are preparing the pre-flight testing review package. o The science verification test plan has been worked on, is continuously updated and anticipated to be finalized in within this funding period. o Flight Mission Patch for MeF1 payload was designed, and is in production.

• Experimental accomplishments: Since the flight hardware selection process was delayed for various reasons, several hardware-dependent science verification experiments could not be performed during the last 10 month. However, since a flight hardware was assigned, such SVT related experiments were initialized. These include, but are not limited to, biocompatibility and toxicity tests of the proposed flight hardware using CD34+ hematopoietic stem cells. In addition, cell seed density and cell survival rate verification tests will be implemented in the next months. Furthermore, experimental testing of microscopy capabilities, including clarity and bubble forming tests will be performed. Finally, the impact of media changes and the implemented media change techniques on cell culture performance will be tested using the flight hardware culture device. The following experiments were accomplished and generated valuable insights and data: • Experiments related to Specific Aim 1: o Experiments using n=6 mobilized adult CD34+ hematopoietic stem cell cultures were performed comparing standard suspension versus rotating wall vessel culture. We were able to demonstrate an appropriate increase in cell numbers, and stable cell viability. Flowcytometric data were analyzed, as well as cell morphology and ability to form proplatelet extension by means of live-microscopy and confocal immuno-cytochemistry. Cells were harvested and submitted to the University of Utah sequencing core facility for further sequencing analysis. o Data gathered from the total RNA sequencing runs are analyzed and verification experiments targeting transcripts of interest will be initiated in the next months. o The experiment simulating galactic cosmic rays (simGCR) was performed using the NSRL facilities at Brookhaven National Laboratory. A rerun of the simGCR experiment (due to anomaly experienced during the initial run) was applied for and will take place in Fall of 2024.

• Experiments related to Specific Aim 2: Several standard ground cell culture conditions need to be adjusted due to in-flight conditions on-board the ISS. o During preliminary experiments, we detected that preserving CD34+ hematopoietic stem cells with RNAlater would result in a dissociation of ribosomes from the respective target RNA sequence, and therefore, making ribosomal protected fragment sequencing impossible. Extensive testing demonstrated that different strategies resulted in preservation of cells providing integrity of ribosome-RNA complexes (using polysome analysis), and furthermore, resulting in RNA integrity numbers (RIN) well within the mission success criteria defined in the SRD. Furthermore, cell cultures used for experiments described in Specific Aim 1 were preserved using such method, and post -sequencing quality control variables demonstrated total read counts and read coverage being within the mission success criteria for excellent scientific return. o On-orbit cell culture experiments will include morphologic studies conducted using live cell imaging approaches. Since the tubulin cytoskeleton is a major contributor to regulated proplatelet formation, we selected different tubulin probes suitable for live microscopy for further testing. All tested live cell imaging dyes can be used without additional washing steps per manufacturer’s protocol, which will significantly reduce astronaut hands-on time. The experimental results demonstrated sufficient staining characteristics of the selected live imaging probes when incubating the cells for different time points. In addition, using a 20x objective, we were able to readily identify detailed tubulin cytoskeletal structures within proplatelet extensions. This test-series was completed, and an appropriate on-orbit live-staining protocol can be implemented. o On-orbit cell culture experiments will require extended fixation of cells using paraformaldehyde (PFA) or alternative approaches. A series of experiments demonstrated that CD34-derived megakaryocytes can be stored for 5 weeks in the appropriate PFA solution at 4?C while preserving morphology and antigenicity. o After identification of the flight hardware, the PI team conducted cell growth / differentiation experiments using the flight hardware materials (as provided by IP). CD34+ hematopoietic stem cells were in direct contact to such materials during the entire culture period, and results were compared to standard lab cell culture conditions. CD34+ hematopoietic stem cells demonstrated tolerance towards being exposed to materials which will be used for the experimental setting on board the ISS. Cells expanded as expected and demonstrated viability percentages comparable to regular culture conditions, even when media changes were performed using syringe and cannula. Furthermore, proplatelet formation was preserved, which was also verified by an unaltered performance of the tubulin live stain.

• Experiments related to Specific Aim 3: The amount of whole blood which can be obtained from the human subjects in Specific Aim 3 is limited, we performed preliminary experiments mimicking the experimental set-up planned to be implemented for Specific Aim 3. o Platelets were isolated from healthy volunteers using whole blood volumes proposed in Specific Aim 3. Harvested numbers of platelets were suitable to perform proposed experiments.

This proposal is based on our robust preliminary data demonstrating that conditions mimicking microgravity (rotating wall vessel culture, RWVC) markedly alter human MK morphology and gene expression. We hypothesize that microgravity will re-program MKs and newly-released PLTs, resulting in critical changes in their transcriptome, proteome, and alterations in PLT number and function. We will determine how microgravity and space radiation conditions on board the International Space Station (ISS) alter human MK and PLT maturation/production, gene expression (DNA, RNA, and protein), and cellular function. We will study in vitro human hematopoietic progenitor cell (HPC)-derived MKs in Earth-based experiments under standard or microgravity conditions. In parallel, human MKs will be studied on the ISS. Integrated, cutting-edge OMICS toolsets (e.g., RNA-sequencing and ribosomal footprinting [Ribo-seq]), comprehensive morphologic studies, and cell production kinetic studies will be used. They will provide unprecedented insight into adaptation processes needed for MK and PLT function under conditions experienced by humans performing spaceflights. These studies will directly address crew health concerns that currently limit human space exploration and will assist in developing targeted countermeasures.

This proposal concurs with the major National Research Council (NRC) Decadal Survey Recommendations for cellular and molecular biology studies using state- of-the-art tools coupled with systems biology, and for studies evaluating the physiological interplay of cardiopulmonary and immune functions during application of spaceflight. Furthermore, we will address goals of the NASA Space Biology Science Plan 2016-2025, including: (1) determine the effects of the space environment on DNA function, (2) develop a systems biology-based understanding of the cellular and molecular changes to explain how gravitational changes in spaceflight effects organisms and causes phenotypic changes, and (3) identify how spaceflight affects the ability of cells to generate and maintain their complex internal cyto-architecture, processes critical for MKs and PLTs.

• ADMINISTRATIVE/ORGANIZATIONAL ACCOMPLISHMENTS: o A flight hardware selection process was initiated by performing a risk-benefit-analysis and creating the required flight concept chart. This process resulted in the conception of flight hardware requirements, which were communicated to the International Space Station National Laboratory (ISSNLP). o The Principal Investigator (PI) and Project Scientist prepared the Science Requirements Document, which was submitted for further review and approval. o The request for beam time to simulate galactic cosmic rays at the NASA Space Radiation Laboratory (NSRL) at the U.S. Department of Energy Brookhaven National Laboratory, was submitted. The requested beam time for the 2023 Summer campaign was granted, the project was discussed with the staff physicists and biologists, and we are waiting for the specific timeslot to be announced. o An Institutional Review Board (IRB) application for the work with mobilized CD34+ hematopoietic stem cells was prepared and submitted to the University of Utah IRB. The project did not meet the definitions of Human Subject Research according to Federal regulations; therefore, IRB oversight is not required or necessary for projects proposed under Specific Aim 1 and 2 of this NASA proposal. o We submitted the NASA IRB application covering Human Subject Research as proposed in Specific Aim 3 of this proposal. The IRB application is currently under review, after passing the pre-review process.

• EXPERIMENTAL ACCOMPLISHMENTS: Since the flight hardware selection process is delayed for various reasons, several hardware-dependent science verification experiments cannot be performed at this time. These include, but are not limited to, biocompatibility and toxicity tests of the proposed flight hardware using CD34+ hematopoietic stem cells. In addition, cell seed density and cell survival rate verification tests can only be implemented once the flight hardware is selected and defined. Furthermore, experimental testing of microscopy capabilities, including clarity and bubble forming tests, will be performed once the flight hardware and accompanying microscopy systems are defined. Finally, the impact of media changes and the implemented media change techniques on cell culture performance can only be tested once the flight hardware is selected. The start of a new post-doctoral fellow also required intensified teaching and training experiments.

The following experiments were accomplished and generated valuable insights and data:

• Experiments related to Specific Aim 1: o In a first experiment, mobilized adult CD34+ hematopoietic stem cells performed as expected during rotating wall vessel culture. We were able to demonstrate an appropriate increase in cell numbers, and stable cell viability around 90%. Furthermore, flow cytometric data showed appropriate expression of differentiation markers (i.e., CD41, CD42b, and CD61). o In a subsequent set of experiments, we studied a multitude of different experimental conditions to define best ground control practice when performing experiments comparing to the rotating wall vessel cell culture approach. Analyzing total cell counts, viability, proplatelet formation capabilities, and flow cytometry we could confidently conclude that using cell culture dishes would be best suited serving as suspension culture control. It is important to note that keeping the cell culture media volume constant during the entire duration of the experiment, independent of the total cell count, best mimics conditions being present in the rotating wall vessel culture dish. This experiment also confirmed our previous findings, demonstrating that CD34+ hematopoietic stem cells depict a larger size when cultured under microgravity simulating conditions.

• Experiments related to Specific Aim 2: Several standard ground cell culture conditions need to be adjusted due to in-flight conditions on board the ISS. o For the proposed flight experiments, we will use pre-mixed cell culture media, already containing growth factors, instead of freshly prepared media formulations used for each media change when conducting standard ground-based experiments. We therefore tested if cell count, viability, and differentiation markers of CD34+ hematopoietic stem cells would change over the course of the experiment due to the use of freshly prepared versus pre-mixed and stored media formulations. We found that cell numbers, viability, and the flow cytometric detection of differentiation markers (i.e., CD41, and CD61) were comparable between freshly prepared and pre-mixed media formulations, indicating that the proposed flight protocol using such pre-mixed cell culture media is appropriate and will not introduce unwanted and inadvertent cell culture effects. o To further evaluate preferred cell culture conditions, we implemented experiments using differential growth factor treatment regimes. Our results demonstrated that stem cell factor is an integral part of successfully culturing CD34+ hematopoietic stem cells. However, results for cell numbers and cell culture viability were comparable and independent of having stem cell factor removed from cell culture media on day 3 or day 6 of culture. Therefore, the proposed in-flight protocol, using stem cell factor and thrombopoietin (TPO) containing media until day 6, and continuing the culture with media containing TPO only until the end of culture period, will be implemented for all cultures. o Due to ISS-introduced limitations, megakaryocytes harvested in orbit cannot be immediately frozen or even further processed towards RNA isolation and subsequent sequencing, as is custom when carrying out ground-based cell studies. Therefore, we tested several methods employing highly efficient preservation of RNA integrity, even if samples need to be stored at room temperature for prolonged periods of time (i.e., two weeks). RNA isolates from cells suspended in RNAlater, and stored at room temperature for two weeks demonstrated the highest degree of integrity, reflected in RNA integrity numbers (RIN) >8. These scores are within the “excellent range” of the Mission Success Criteria defined by the Science Requirements Document draft. o On-orbit cell culture experiments will include morphologic studies conducted using live cell imaging approaches. Since the tubulin cytoskeleton is a major contributor to regulated proplatelet formation, we selected different tubulin probes suitable for live microscopy for further testing. All tested live cell imaging dyes can be used without additional washing steps per manufacturer’s protocol, which will significantly reduce astronaut hands-on time. The experimental results demonstrated sufficient staining characteristics of the selected live imaging probes when incubating the cells for 30 minutes. In addition, using a 20x objective, we were able to readily identify detailed tubulin cytoskeletal structures within proplatelet extensions. o To address potential interference of the aforementioned cellular live stain with RNA isolation procedures, we performed experiments combining the tubulin probes for live microscopy with subsequent RNA isolation techniques. This approach enabled us to optimize established protocols. Using RNAlater as cell preservative, we found that using the tubulin live stain did not result in loss of RNA. Furthermore, RNA integrity numbers were >8, falling well within the excellent range of the Mission Success Criteria defined by the Science Requirements Document draft.

This proposal is based on our robust preliminary data demonstrating that conditions mimicking microgravity (rotating wall vessel culture, RWVC) markedly alter human MK morphology and gene expression. We hypothesize that microgravity will re-program MKs and newly-released PLTs, resulting in critical changes in their transcriptome, proteome, and alterations in PLT number and function. We will determine how microgravity and space radiation conditions on board the International Space Station (ISS) alter human MK and PLT maturation/production, gene expression (DNA, RNA, and protein), and cellular function. We will study in vitro human hematopoietic progenitor cell (HPC)-derived MKs in Earth-based experiments under standard or microgravity conditions. In parallel, human MKs will be studied on the ISS. Integrated, cutting-edge OMICS toolsets (e.g., RNA-sequencing and ribosomal footprinting [Ribo-seq]), comprehensive morphologic studies, and cell production kinetic studies will be used. They will provide unprecedented insight into adaptation processes needed for MK and PLT function under conditions experienced by humans performing spaceflights. These studies will directly address crew health concerns that currently limit human space exploration and will assist in developing targeted countermeasures.

This proposal concurs with the major National Research Council (NRC) Decadal Survey Recommendations for cellular and molecular biology studies using state- of-the-art tools coupled with systems biology, and for studies evaluating the physiological interplay of cardiopulmonary and immune functions during application of spaceflight. Furthermore, we will address goals of the NASA Space Biology Science Plan 2016-2025, including: (1) determine the effects of the space environment on DNA function, (2) develop a systems biology-based understanding of the cellular and molecular changes to explain how gravitational changes in spaceflight effects organisms and causes phenotypic changes, and (3) identify how spaceflight affects the ability of cells to generate and maintain their complex internal cyto-architecture, processes critical for MKs and PLTs.