iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications

Abstract

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

Keywords: cold adaptation; ground squirrel; hibernation; hypothermia; induced pluripotent stem cells; lysosomal membrane permeabilization; microtubule cold stability; mitochondria; organ storage; retina.

Figure 1. Species-dependent Differences in Neuronal Microtubule Cold Stability

(A) Immunofluorescence of TUBB3 (red), polyglutamylated tubulin (poly-E-T; green), or delta2-tubulin (Δ2-T; blue) in cultured rat primary cortical neurons incubated at 4°C for various durations. Scale bar: 40 μm. (B) Immunofluorescence of TUBB3, poly-E-T, or Δ2-T in cultured human induced pluripotent stem cell-derived neurons (iPSC-neurons) incubated at 4°C for various durations. Scale bar: 40 μm. (C) Immunofluorescence of TUBB3, poly-E-T, or Δ2-T in cultured 13-lined ground squirrel (GS) primary cortical neurons incubated at 4°C for various durations. Scale bar: 40 μm. (D) Immunofluorescence of TUBB3, poly-E-T, or Δ2-T in GS iPSC-neurons incubated at 4°C for various durations. Scale bar: 40 μm. For other cell types differentiated from GS iPSCs, see also Figure S1. (E) Cumulative plots and quantification of the lengths of manually traced TUBB3-positive microtubules (see STAR METHODS) from GS cortical primary neurons (n = 5 experiments each at 37°C and 4°C; cultures derived from 4 GS pups) and GS iPSC-neurons (n = 6 experiments each at 37°C and 4°C; cultures derived from 2 GS iPSC lines). Cumulative lengths of short (neurite lengths < 50% of the cumulative plots) and long microtubules (neurite lengths > 50% of the cumulative plots) were quantified separately. No significant difference in microtubule length attributed to cold temperature was found (p > 0.05; Student’s t-test for two-group comparison).

Figure 2. Species-dependent iPSC-neuronal Transcriptome Changes in Mitochondria and Protein Quality Control (PQC) in Response to Low Temperature

(A) Western blots and quantification of TUBB3, poly-E-T and Δ2-T proteins in GS and human iPSC-neurons following 4- or 16-h cold exposure with or without TUBB3 overexpression (n = 5 experiments from 2 GS and 2 human cell lines; Student’s t-test between untreated controls with or without cold exposure, and between cold-exposed groups with or without the TUBB3 overexpression plasmid; ** p < 0.01; *** p < 0.001). See also Figure S2C. (B) Heatmaps of transcriptomic alterations in GS and human iPSC-neuronal cultures induced by 4°C incubation for 1 h or 4 h (see STAR METHODS), highlighting two distinct categories of expression- genes related to mitochondrial functions, and genes encoding heat shock proteins (HSPs), proteases and proteinase inhibitors (SERPINs, magenta). Some genes exhibiting distinct alteration patterns between GS and human are emphasized. See also Figure S3, Data Files S1–S3.

Figure 3. Cold-induced Mitochondrial Hyperpolarization and Oxidative Stress in Human iPSC-neurons

(A) Live cell imaging protocol (left) of mitochondrial membrane potential (Δψ_m) with TMRE (see STAR METHODS), and quantification (right) of cold-induced Δψ_m changes in GS and human iPSC-neurons untreated controls, and treated with BAM15 or PI (n = 8 experiments; Student’s t-test between GS and human controls; ANOVA plus post hoc Tukey test for multiple human cold-exposed groups with or without drug treatment; *** p < 0.001; n.s. p > 0.05, not significant). BAM15: a mitochondrial uncoupling drug; PI: protease inhibitors. See also Movie S1. (B) CellROX green imaging protocol (left) to assess cold-induced ROS production (see STAR METHODS), and quantification (right) of ROS production following 30-min cold exposure in GS and human iPSC-neurons (n = 8 experiments; Student’s t-test between GS and human comparisons; ANOVA plus post hoc Tukey test for multiple human cold-exposed groups with or without drug treatment; ** p < 0.01; n.s. p > 0.05, not significant). (C) Western blots and quantification of oxidized proteins following 16-h incubation at 4°C (n = 5 experiments; Student’s t-test for two-group comparisons; * p < 0.05). (D) Immunofluorescence of TUBB3 (magenta) and oxidized proteins (green). Note: after 16-h incubation at 4°C, minimal protein oxidation was detected on GS TUBB3+ processes even with digital enhancement, while in human neurons oxidized proteins were found on residual TUBB3+ microtubules, which could be reduced or completely mitigated by BAM15 treatment (n = 5 experiments). Scale bar: 50 μm. See also Figure S4. For all experiments in this figure, neurons were derived from 2 GS and 3 human iPSC lines for each group.

Figure 4. Cold-induced Lysosomal Membrane Permeabilization (LMP) in Human iPSC-neurons

(A) (Left panel) Live imaging of lysosomes in GS and human iPSC-neurons with DND-26 (green) and Magic Red (magenta; see STAR METHODS). Note: Following 1-h incubation at 4°C, accumulation of diffuse cytoplasmic fluorescence in marked human neurons (arrowheads) indicating LMP. Scale bar: 20 μm. (Right Panel) Quantification of cells containing diffused Magic Red signals (Diameter > 5μm) (n = 6 experiments; Student’s t-test for two-group comparison; *** p < 0.001). (B) (Left panel) Live imaging of lysosomes in human iPSC-neurons with Magic Red under denoted conditions. Note: BAM15 (0.1 μM) or the antioxidant vitamin C (0.5 mM) alleviated LMP in human neurons treated following 4-h incubation at 4°C. Scale bar: 20 μm. (Right Panel) Quantification of cells containing diffused Magic Red signals (Diameter > 5μm) (n = 5 experiments; Student’s t-test between untreated controls with or without cold exposure; ANOVA plus post hoc Tukey test for multiple cold-exposed groups; ** p < 0.01; *** p < 0.001). See also Figure S5 for the involvement of other PQC components. For these experiments, neurons were derived from 2 GS and 3 human iPSC lines for each group.

Figure 5. Morphological Protection of Human iPSC-neurons by BAM15/PI Pretreatments against Prolonged Cold Stress

(A) Pre-treatment with BAM15 (0.1 μM; n = 17 experiments), PI (1:500; n = 24 experiments) or a combination of both (n = 20 experiments) preserved long neurites (poly-E-T: green; TUBB3: red; Δ2-T: blue) of human iPSC-neurons following 4-h incubation at 4°C. Scale bar: 40 μm. See also Figure S6. (B) Cumulative plots and quantification of TUBB3+ neurite lengths (see STAR METHODS; BAM15: n = 6 experiments; PI: n = 5 experiments; BAM15 & PI: n = 6 experiments; Student’s t-test between untreated controls with or without cold exposure; ANOVA plus post hoc Tukey test for multiple cold-exposed groups treated for the same duration; * p < 0.05; ** p < 0.01; *** p < 0.001). (C) Western blots and quantification confirming that BAM15 or PI pre-treatment maintained tubulin protein levels in human iPSC-neurons after 4-h incubation at 4°C (n = 5 experiments; Student’s t-test between untreated controls with or without cold exposure; ANOVA plus post hoc Tukey test for multiple cold-exposed groups; ** p < 0.01; *** p < 0.001). (D) Proposed model depicting the mechanisms by which BAM15 and PI pre-treatments protect human iPSC-neurons from cold stress. For these experiments, human neurons were derived from 3 iPSC lines.

Figure 6. Morphological and Functional Protection of Rat Retinal Explants by BAM15/PI Pretreatments against Prolonged Cold Stress

(A) Microtubule morphology in rat (n = 10 animals) retinal ganglion cells (RGCs) immunostained for TUBB3 (red). Scale bars: 20 μm. (B) Cumulative plots and quantification of TUBB3+ RGC dendritic lengths in rat retina (see STAR METHODS; n = 6 animals; Student’s t-test for two-group comparisons; ** p < 0.01; *** p < 0.001). (C) Quantification of multielectrode array (MEA) recordings of spontaneous RGC activity following exposure to 4°C for 0 h, 4 h, or 24 h. Treatments and their corresponding color legend are indicated. Upper-left panel: Numbers of active RGCs detected by MEA. Upper-right panel: Average firing rates for detected RGCs. Lower panel: Distributions of firing rates for detected RGCs. Student’s t-test between untreated controls with or without cold exposure, and the 24-h cold-exposed groups; ANOVA plus post hoc Tukey test for multiple cold-exposed groups treated for the same duration; * p < 0.05; ** p < 0.01; *** p < 0.001. See also Movie S2 for an animated example of MEA recordings of the RGC spontaneous firing. The number of animals used in each condition is provided in the histogram. (D) Representative light responses of rat RGCs following 4-h cold exposure either untreated or treated with BAM15 (n = 5 animals), PI (n = 6 animals), or Taxol (n = 6 animals). Quantification is provided as the percentage of detected RGCs that were light-responsive (see STAR METHODS; Student’s t-test between untreated controls with or without cold exposure; ANOVA plus post hoc Tukey test for multiple groups cold-exposed for the same duration; * p < 0.05; *** p < 0.001). See also Movie S3 for an animated example of MEA recordings of the light response experiments.

Figure 7. Protecting Mouse Kidneys with BAM15/PI during Cold Storage

Immunofluorescence of α-tubulins (TUBA; green) and DAPI (magenta) in transverse sections of wild-type mouse kidneys under these conditions (n = 4–6 animals used for each condition): freshly fixed, after 24-h cold storage at 4°C (with or without BAM15/PI) or rewarming at 37°C (with or without BAM15/PI) for 30 min following 24-h cold storage. Note: Reduced tubulin signals in glomeruli (arrowheads in upper panel; middle panel) and renal tubular epithelium (lower panel) of mouse kidneys stored at 4°C for 24 h in standard University of Wisconsin (UW) Solution. Scale bars: 25 μm in all panels. See also Figure S7 for oxidative damages and cell apoptosis in mouse kidneys during cold storage.