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.

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.

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.