Human Neural Stem Cells Flown into Space Proliferate and Generate Young Neurons

Abstract

Here we demonstrate that human neural stem cells (NSCs) proliferate while in space and they express specific NSC markers after being in space. NSCs displayed both higher oxygen consumption and glycolysis than ground controls. These cells also kept their ability to become young neurons. Electrophysiological recordings of space NSC-derived neurons showed immature cell membrane properties characterized by small capacitance and very high input resistance. Current injections elicited only an incipient action potential. No spontaneous synaptic events could be detected, suggesting their immature status even though most recorded cells displayed complex morphology and numerous cell processes. Ascertaining the origin of the NSCs' increased energy requirement is of the essence in order to design effective measures to minimize health risks associated with long-duration human spaceflight missions.

Keywords: energetics; glycolysis; microgravity; neural stem cells; neuronal specification; neurons; pluripotency; proliferation; space flight.

Figure 1.

(A) View of an automated Type IV Unit Kiwi (Airbus). (B) Tank 1 (green) contained NSCs, medium, and BrdU. At time (T) + 2 this medium was delivered to NSCs, at T + 5 medium from the second tank (blue) was delivered to arrest the cells and the units were placed at 4 °C. Two units containing 12 pieces of mesh carrier with cells were used for the “in space” proliferation study (C) Representative view of the mesh carrier on which tire NSCs were seeded. (D) Mesh with NSCs recovered from the devices post-flight. (E,F) Views of StaARS-1, which offers real-time command capability without the need for astronaut time.

Figure 2.. Human NSCs proliferate in space.

Triple immunofluorescence performed on cells adhered to the mesh-cell carrier. A to D show NSCs that proliferated while in space. E to H show views of ground control NSCs. (A) The PSA-NCAM antibody labeled most but not all cells. (B) Cells expressing nestin as seen by the intensity of the marker in all NSCs. (C) Different intensities of BrdU-labeled cells, some organized in rows (arrowheads) or as dividing cells (arrows) as shown in the insets. (D) Most cells expressed BrdU and nestin and a subpopulation colocalized with PSA-NCAM. (E) PSA-NCAM was not expressed by the NSCr grown in 1 G (ground control). (F) Some cells expressed nestin at different intensities and others were negative. (G) Only some cells intensely expressed BrdU (arrowheads), while most had a faint expression (open arrowheads) or were negative. (H) The merged image shows that there were less cells in 1 G (ground controls) with respect to the cells sent to space. The BrdU incubation was for 72 h for all cells in automated units. (Bar = 50 μm).

Figure 3.

SPC-NSCs and ground control NSCs were recovered from the passive hardware (A) and treated in the same manner. (B) Representative example of past-flight NSCs that continued to actively proliferate, as shown by the arrows. (C) After 39.3 days in space, NSCs display enhanced energy consumption. OCR—oxygen consumption rate; ECAR—extracellular acidification rete; SPC-NSCs in the space environment. Each experiment consisted of n = 12. The values are expressed as mean + SD. * The Student’s t test revealed that these differences were significant (p < 0.05).

Figure 4.. SPC-NSCs gave rise to neurons after space flight.

(A)—(left panels) Whole-cell voltage clamp recording; using; K-gluconate in the internal solution: (top) step voltage commands from −80 to +10 mV elicited only minimal inward currents, whereas outward currents were predominant, and (bottom) graph represents an IV relationship, illustrating the presence of large outward currents starting at −30 mV. (B) When the recording was switched to current clamp mode, small step current pulses evoked large negative voltage deflections and only an incipient action potential with depolarizing current injections, demonstrating a neuronal phenotype, yet still very immature. (C) Infrared video-microscopy images of human neurons derived from NSCs flown into space. The top left panel shows a cluster of cells with multiple processes emanating from different cells. The top right panel shows an NSC with bipolar morphology. The bottom left panel shows the same cell after patch and electrophysiological recordings (illustrated in A and B) were obtained. The bottom right panel shows another NSC after patching.

Scheme 1.

Timeline 1: This diagram illustrates the times at which culture media changes took place while neural stem cells (NSCs) traveled and were in space in the automated hardware at 37 °C. Culture medium from Tank 1 containing bromodeoxyuridine (BrdU) was released into the cell chamber 2 days after docking. Three days later (at 5 days of having been in space), the second medium without BrdU was released and the units were stored at 4 °C. Proliferation while in space was ascertained during only 3 days.

Scheme 2.

Timeline 2.