Placenta-Expanded Stromal Cell Therapy in a Rodent Model of Simulated Weightlessness

Abstract

Long duration spaceflight poses potential health risks to astronauts during flight and re-adaptation after return to Earth. There is an emerging need for NASA to provide successful and reliable therapeutics for long duration missions when capability for medical intervention will be limited. Clinically relevant, human placenta-derived therapeutic stromal cells (PLX-PAD) are a promising therapeutic alternative. We found that treatment of adult female mice with PLX-PAD near the onset of simulated weightlessness by hindlimb unloading (HU, 30 d) was well-tolerated and partially mitigated decrements caused by HU. Specifically, PLX-PAD treatment rescued HU-induced thymic atrophy, and mitigated HU-induced changes in percentages of circulating neutrophils, but did not rescue changes in the percentages of lymphocytes, monocytes, natural killer (NK) cells, T-cells and splenic atrophy. Further, PLX-PAD partially mitigated HU effects on the expression of select cytokines in the hippocampus. In contrast, PLX-PAD failed to protect bone and muscle from HU-induced effects, suggesting that the mechanisms which regulate the structure of these mechanosensitive tissues in response to disuse are discrete from those that regulate the immune- and central nervous system (CNS). These findings support the therapeutic potential of placenta-derived stromal cells for select physiological deficits during simulated spaceflight. Multiple countermeasures are likely needed for comprehensive protection from the deleterious effects of prolonged spaceflight.

Keywords: CNS; PLX-PAD cells; bone loss; cell therapy; cytokines; hindlimb unloading; hippocampus; immune system; muscle atrophy; placenta; spaceflight.

Figure 1

Study design and timing of procedures. (a) Timeline of treatments and procedures in this study. Hindlimb unloading (HU) and normally loaded (NL, control) groups received two intramuscular injections of PLX-PAD or PlasmaLyte (Sham) and euthanized 30 days after onset of HU. (b) Summary of experimental groups.

Figure 2

Body weights of experimental groups and measures of neuroendocrine stress. (a) Body weights from acclimation (Day −3) to Day 30 of hindlimb unloaded (HU) mice and normally loaded (NL) controls. Repeated measures ANOVA was performed. + Statistically significant difference between NL Sham vs. HU Sham at p < 0.05; * Statistically significant difference between NL Sham vs. HU Sham and NL PAD vs. HU PAD at p < 0.05. Sample sizes are: NL Sham (n = 12), NL PAD (n = 12), HU Sham (n = 11), and HU PAD (n = 9). Statistically significant at p < 0.05 by one-way ANOVA with Tukey post-hoc test. (b) Plasma corticosterone levels at Day 30 post-HU. NL Sham (n = 11), NL PAD (n = 12), HU Sham (n = 11), and HU PAD (n = 10). A nonparametric Wilcoxon all pairs test was performed; statistically significant at p < 0.05. (c) Total weights of left and right adrenals normalized to body weight. ‘PAD’ denotes PLX-PAD administration. Sample sizes are: NL Sham (n = 12), NL PAD (n = 12), HU Sham (n = 9), and HU PAD (n = 9). Statistically significant at p < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 3

Musculoskeletal measurements. (a,b) μCT analysis of mouse tibiae. (a) Percent cancellous bone volume (BV/TV, %). NL Sham (n = 12), NL PAD (n = 12), HU Sham (n = 10), and HU PAD (n = 10). * Statistically significant at p < 0.05 by nonparametric Wilcoxon all pairs test at p < 0.05. (b) Cortical thickness (Ct.Th.). NL Sham (n = 11), NL PAD (n = 12), HU Sham (n = 11), and HU PAD (n = 10). * Statistically significant at p < 0.05 by one-way ANOVA with Tukey post-hoc test. (c) Right soleus weight normalized to body weight. NL Sham (n = 12), NL PAD (n = 12), HU Sham (n = 11), and HU PAD (n = 9). * Statistically significant at p < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 4

Immune organ weight and cell profiling at day 30 post-HU. (a) Thymus and (b) spleen weights normalized to body weights at day 30 post-HU. (c–f) Results from flow cytometry of whole blood collected from mice at day 30 post-HU. (c) Neutrophil within leukocytes population percentages (%, Ly6g+CD11b+/CD45+), (d) Lymphocytes within leukocyte population percentage (%, CD11b−/CD45+), (e) Neutrophil to lymphocyte ratio (NLR), and (f) Total monocyte to leukocyte population percentage (%, CD11b+/CD45+). NL Sham (n = 12), NL PAD (n = 12), HU Sham (n = 11), and HU PAD (n = 9). * Statistically significant at p < 0.05 by one-way ANOVA with Tukey post-hoc test.

Figure 5

Protein levels of representative cytokines in hippocampus normalized to total protein content at day 30 post-HU. (a) IL-2, (b) IL-6 and (c) M-CSF and (d) CXCL-9. NL Sham (n = 8), NL PAD (n = 6), HU Sham (n = 8), and HU PAD (n = 8). * Statistically significant by one-way ANOVA and Tukey post-hoc test at p < 0.05.