The tardigrade-derived mitochondrial abundant heat soluble protein improves adipose-derived stem cell survival against representative stressors

Abstract

Human adipose-derived stem cell (ASC) grafts have emerged as a powerful tool in regenerative medicine. However, ASC therapeutic potential is hindered by stressors throughout their use. Here we demonstrate the transgenic expression of the tardigrade-derived mitochondrial abundant heat soluble (MAHS) protein for improved ASC resistance to metabolic, mitochondrial, and injection shear stress. In vitro, MAHS-expressing ASCs demonstrate up to 61% increased cell survival following 72 h of incubation in phosphate buffered saline containing 20% media. Following up to 3.5% DMSO exposure for up to 72 h, a 14-49% increase in MAHS-expressing ASC survival was observed. Further, MAHS expression in ASCs is associated with up to 39% improved cell viability following injection through clinically relevant 27-, 32-, and 34-gauge needles. Our results reveal that MAHS expression in ASCs supports survival in response to a variety of common stressors associated with regenerative therapies, thereby motivating further investigation into MAHS as an agent for stem cell stress resistance. However, differentiation capacity in MAHS-expressing ASCs appears to be skewed in favor of osteogenesis over adipogenesis. Specifically, activity of the early bone formation marker alkaline phosphatase is increased by 74% in MAHS-expressing ASCs following 14 days in osteogenic media. Conversely, positive area of the neutral lipid droplet marker BODIPY is decreased by up to 10% in MAHS-transgenic ASCs following 14 days in adipogenic media. Interestingly, media supplementation with up to 40 mM glucose is sufficient to restore adipogenic differentiation within 14 days, prompting further analysis of mechanisms underlying interference between MAHS and differentiation processes.

Keywords: Injections; Stem cells; Stress tolerance; Tardigrade; Transgenic.

Figure 1

Mitochondrial localization of MAHS. (A) Quantification of Pearson correlation coefficient for the overlap between expressed protein localization and mitochondrial morphology (two-sample t-test, n = 4). The denoted p-value indicates the pairwise difference in GFP/MitoView correlation between the genotypes. (B) Maximum projections of the overlap between expressed protein localization (AcGFP1 or AcGFP1-MAHS, cyan) and mitochondrial morphology (MitoView, yellow) in ASC52telos transduced with either the AcGFP1 or AcGFP1-MAHS transgene. Scale bars are 25 µm.

Figure 2

DMSO stress. (A) Maximum projections of the nuclear (DAPI, cyan) and cytoskeletal (phalloidin, magenta) structures of ASC52telos transduced with either the AcGFP1 or MAHS transgene following 72 h of DMSO treatment. Scale bars are 200 µm. (B) Quantification of cell population in AcGFP1- and MAHS-expressing ASCs following a 72 h DMSO treatment (ANCOVA, Tukey’s HSD, n = 3). Cell population quantification was normalized to cells cultured without DMSO for 72 h. Denoted p-values indicate pairwise difference in cell population between the genotypes at each specific DMSO concentration.

Figure 3

Injection shear stress. (A) Maximum projections of live (Calcein-AM, yellow) and dead (EthD-III, magenta) AcGFP1- and MAHS-expressing ASC52telos following injection through various needle gauges. Scale bars are 300 µm. (B) Quantification of cell survival in AcGFP1- (cyan) and MAHS-transgenic (magenta) ASCs following injection through various needle gauges (2-way ANOVA, Tukey’s HSD, n = 6). Denoted p-values indicate pairwise difference in cell survival between the genotypes at each specific needle gauge.

Figure 4

Metabolic stress. (A) Maximum projections of the nuclear (DAPI, cyan) and cytoskeletal (phalloidin, magenta) structures of ASC52telos transduced with either the AcGFP1 or MAHS transgene following 72 h of media depletion. Scale bars are 300 µm. (B) Quantification of cell population in AcGFP1- and MAHS-expressing ASCs following a 72 h media depletion (ANOVAN, Tukey’s HSD, n = 3). Cell survival quantification was normalized to cells cultured in 100% media for 72 h. Denoted p-values indicate pairwise difference in cell population between the genotypes at each specific media concentration.

Figure 5

Osteogenic differentiation. (A) Brightfield images of 14 day alkaline phosphatase expression (ALP, scale bars = 300 µm) in AcGFP1- and MAHS-transgenic ASCs. (B) Quantification of 14 day ALP expression (two-sample t-test, n = 3). The denoted p-value indicates the pairwise difference in ALP expression between the genotypes.

Figure 6

Adipogenic differentiation. (A) Maximum projections of 14 day BODIPY positive area (BODIPY, scale bars = 250 µm) in AcGFP1- and MAHS-transgenic ASCs. (B) Quantification of 14 day BODIPY positive area (two-sample t-test, n = 3). Denoted p-values indicate pairwise difference in BODIPY positive area between the genotypes at each specific time point.

Figure 7

Adipogenic differentiation (14 day glucose-treated basal and adipogenic media). (A) Maximum projections of 14 day BODIPY positive area (BODIPY, scale bars = 250 µm) in AcGFP1- and MAHS-transgenic ASCs following culture in glucose-treated basal and adipogenic media. (B) Quantification of 14 day BODIPY positive area in glucose-treated basal and adipogenic media in AcGFP1- and MAHS-expressing ASCs (ANOVAN, Tukey’s HSD, n = 3). Denoted p-values indicate pairwise difference in BODIPY positive area between the genotypes at each specific glucose concentration.

Figure 8

Adipogenic differentiation (21 day glucose-treated basal and adipogenic media). (A) Maximum projections of 21 day BODIPY positive area (BODIPY, scale bars = 250 µm) in AcGFP1- and MAHS-expressing ASCs following culture in glucose-treated basal and adipogenic media. (B) Quantification of 21 day BODIPY positive area in glucose-treated basal and adipogenic media in AcGFP1- and MAHS-transgenic ASCs (ANOVAN, Tukey’s HSD, n = 3). Denoted p-values indicate pairwise difference in BODIPY positive area between the genotypes at each specific glucose concentration.