NOTE: Extended to 1/31/2022 per F. Hernandez/ARC (Ed., 7/27/21)

NOTE: Extended to 6/30/2021 per F. Hernandez/ARC (Ed., 1/18/21)

NOTE: Extended to 10/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/18/20)

NOTE: Extended to 6/30/2020 per NSSC information (Ed., 1/29/2020)

NOTE: Extended to 11/1/2019 per F. Hernandez/ARC (Ed., 11/6/18)

NOTE: Extended to 11/1/2018 per F. Hernandez/ARC (Ed., 10/21/16)

We are using magnetic levitation, a unique ground-based simulation of orbital free fall, to augment and compare the findings from the Spaceflight Experiment Phase (micro-15). Unfortunately astronaut handling of the samples aboard the ISS resulted in only a few EB’s returned rather than the expected hundreds of EB’s which resulted in a loss of science. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates via magnetic levitation. Using iPS cells derived from the Oct4:CreER mTmG mice and timed tamoxifen additions, it is possible over time to distinguish cells still expressing Oct4 (i.e., expressing GFP), from those that have lost Oct4 expression (expressing RFP). We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

• The gene Oct4 is a key marker of mammalian pluripotency. • The University of Minnesota UMN Oct4CreER::mTmG mouse iPSC line is the best tested, most sensitive Oct4 lineage tracing system currently available and was used for International Space Station (ISS) and ground-based microgravity simulation studies. • Magnetic levitation was used as a unique ground-based simulation of in-orbit microgravity. • Changes in the dynamics of Oct4 loss in simulated and actual microgravity was observed, indicating there are fundamental effects of the space environment on the regulation of this key gene. • This was the first project to employ ISS crew to accomplish media exchange following cell centrifugation – a standard laboratory technique that can now be used in many other ISS experiments. • This research has pioneered the self-assembly of stem cell embryoid bodies (EBs) that can be used by many other future ISS projects using cell aggregate and organoids.

Progress during the current No Cost Extension period:

1) We are now preparing uniform-sized EBs, which will remove uncertainties regarding rate of differentiation, potential oxygen, and nutrient disparities as a function of EB size. The EBs are grown in EZSPHERE™ 35 mm Dishes [Diameter: 500µm, Depth: 200µm, No. of Well: 2,700/dish]. This is compatible with our sample positioning apparatus in the magnetic levitation system. 2) Optimized green fluorescent protein (GFP) and red fluorescent protein (RFP) staining protocols for EBs. This improves quantification of red and green fluorescing cells. 3) Coordinated with University of Minnesota Genomics Center for using its expertise in executing and analyzing genomic data from anticipated studies. Purchased the necessary reagents for spatial genomic analysis of EBs for 1g and simulated microgravity studies. 4) Due to a national liquid helium shortage, we are unable to secure liquid helium for the levitation magnet, which is primarily due to downtime in a helium extraction facility. The expectation is that the shortage will subside during calendar year 2023. We are planning to relocate the maglev system in early 2023 to the NASA Kennedy Space Center (KSC) microgravity simulation facility where helium supplies are anticipated to be less restrictive. This should enable us to utilize the maglev system to complete the science objectives of the grant.

Future work scope and project objectives:

Aim 1: Determine the effect of simulated microgravity, via magnetic levitation, on the timing and spatial arrangement of Oct4 expression in differentiating embryoid bodies /induced pluripotent stem cells (EBs/iPSC) aggregates. - Confocal microscopy will be used to determine pluripotency. Cells will be green if Oct4 is expressing and red if Oct4 is not expressing.

Aim 2: Determine the effect of simulated microgravity, via magnetic levitation, on downstream gene expression in the cohorts of Oct4-expressing and non-expressing cells during cell differentiation. -Spatial genomics (Nanostring GEOMX Digital Spatial Profiling (DSP)) will be used to analyze transcriptomics.

NOTE: Extended to 1/31/2022 per F. Hernandez/ARC (Ed., 7/27/21)

NOTE: Extended to 6/30/2021 per F. Hernandez/ARC (Ed., 1/18/21)

NOTE: Extended to 10/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/18/20)

NOTE: Extended to 6/30/2020 per NSSC information (Ed., 1/29/2020)

NOTE: Extended to 11/1/2019 per F. Hernandez/ARC (Ed., 11/6/18)

NOTE: Extended to 11/1/2018 per F. Hernandez/ARC (Ed., 10/21/16)

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

• The gene Oct4 is a key marker of mammalian pluripotency.

• The UMN Oct4CreER::mTmG mouse iPSC line is the best tested, most sensitive Oct4 lineage tracing system currently available and was used for ISS and ground-based microgravity simulation studies.

• Magnetic levitation was used as a unique ground based simulation of on-orbit microgravity.

• Changes in the dynamics of Oct4 loss in simulated and actual microgravity was observed indicating there are fundamental effects of the space environment on the regulation of this key gene.

• This is the first project to employ ISS crew to accomplish media exchange following cell centrifugation -- a standard laboratory technique that can now be used in many other ISS experiments.

• This research has pioneered the self-assembly of stem cell embryoid bodies (EBs) that can be used by many other future ISS projects using cell aggregate and organoids.

NOTE: Extended to 10/31/2020 per F. Hernandez/ARC and NSSC information (Ed., 6/18/20)

NOTE: Extended to 6/30/2020 per NSSC information (Ed., 1/29/2020)

NOTE: Extended to 11/1/2019 per F. Hernandez/ARC (Ed., 11/6/18)

NOTE: Extended to 11/1/2018 per F. Hernandez/ARC (Ed., 10/21/16)

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

b) Completed SVT (Science Verification Test).

The first phase of the project focused on generating embryoid bodies (EBs), which are 200 micron spherical aggregates of iPSCs (induced Pluripotent Stem Cells) and developing freezing protocols so that EBs can be transported and stored at -95 degrees C aboard the ISS until crew time is allocated to the project. We determined that EBs did not cryopreserve well and investigated the possibility of previously frozen stem cells self-assembling into EBs aboard the ISS. This procedure has been done successfully in a conventional incubator (1g) and when exposed to simulated microgravity via magnetic levitation in our laboratory. These self-aggregated EBs exhibit the same fluorescence patterns when tamoxifen treated as those grown on 96 well plates. A significant benefit of transporting frozen stem cells to the ISS for self-assembly avoids g-force and vibration of launch as well as ground and berthing delays which can damage fragile stem cells.

c) Completed EVT (Experiment Verification Test).

d) Shipped experimental samples to Kennedy Space Center (KSC) 07-15-2019.

e) Launched experiment on 07-25-2019 to ISS on SpaceX CRS-18. Berthing occurred on 07-27-2019.

f) ISS samples returned on 08-29-2019 to University of Minnesota for analysis.

g) Samples were processed/analyzed. Less than 10 EBs recovered from a few culture bags. Most culture bags had zero embryoid bodies (EBs) formed. No evidence of single cells or aggregated cells in most of cell culture bags indicating that cells were inadvertently removed/lost during sample handling on-orbit.

h) Asynchronous ground control commenced in November 2019. iPSCs frozen at -80 degrees C in July 2019 were thawed, cryopreservation media was removed, fresh media was added to sample bags, and allowed to spontaneously form EBs. Excellent yield of embryoid bodies (100s per bag), indicating that samples sent to ISS should have been viable after thaw from frozen state. This reinforces the conclusion that poor sample handling aboard the ISS resulted in the inadvertent loss of cells leading to a failed on-orbit experiment.

i) Requested no-cost-extension to extend to August 30, 2020 to allow for ground-based microgravity simulation studies of EBs in a magnetic levitation system.

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.

During the Flight Definition Phase we will use magnetic levitation, a unique ground-based simulation of orbital free fall, to optimize execution of the Spaceflight Experiment Phase. This approach will maximize the success of space-based studies. We propose to investigate the effect of gravity on the timing and spatial arrangement of the loss of Oct4 expression in differentiating iPS cell aggregates. We will also examine the effect of gravity on gene expression in cohorts of Oct4 expressing and non-expressing cells during differentiation by comparing the results of ground-based experiments to those conducted on orbit. Finally, we will explore mechanisms behind the effect of microgravity on both Oct4 gene regulation and control of downstream gene expression by Oct4. This work will determine the effect of spaceflight on changes in Oct4 gene expression during differentiation of pluripotent stem cells and the consequences of these changes on differentiation outcomes. This will increase our understanding of fundamental stem cell behavior in microgravity.