Evolution of T cells in the cancer-resistant naked mole-rat.

Naked mole-rats (NMRs) are best known for their extreme longevity and cancer resistance, suggesting that their immune system might have evolved to facilitate these phenotypes. Natural killer (NK) and T cells have evolved to detect and destroy cells infected with pathogens and to provide an early response to malignancies. While it is known that NMRs lack NK cells, likely lost during evolution, little is known about their T-cell subsets in terms of the evolution of the genes that regulate their function, their clonotypic diversity, and the thymus where they mature. Here we find, using single-cell transcriptomics, that NMRs have a large circulating population of γδT cells, which in mice and humans mostly reside in peripheral tissues and induce anti-cancer cytotoxicity. Using single-cell-T-cell-receptor sequencing, we find that a cytotoxic γδT-cell subset of NMRs harbors a dominant clonotype, and that their conventional CD8 αßT cells exhibit modest clonotypic diversity. Consistently, perinatal NMR thymuses are considerably smaller than those of mice yet follow similar involution progression. Our findings suggest that NMRs have evolved under a relaxed intracellular pathogenic selective pressure that may have allowed cancer resistance and longevity to become stronger targets of selection to which the immune system has responded by utilizing γδT cells.

Effects of erythropoietin in neonates with hypoxic-ischemic encephalopathy receiving therapeutic hypothermia.

BACKGROUND: Minimizing multiple organ dysfunction-related mortality and morbidity is a critical issue for patients with hypoxic-ischemic encephalopathy (HIE) receiving therapeutic hypothermia (TH). Although erythropoietin (EPO) has demonstrated protective effects on various hypoxic-ischemic organs in animal studies and clinical trials in adults, its effects on neonates with HIE require further investigation. METHODS: This study retrospectively analyzed the medical records of neonates with HIE who received TH with or without EPO (TH+EPO vs TH groups) administration in a tertiary referral hospital from January 2016 to January 2021. Data regarding patient characteristics, medical treatment, and clinical (neurological, cardiac, respiratory, gastrointestinal, hepatic, and renal) function assessments were collected. To control for confounding factors and selection bias between the two groups, a 1:1 propensity matching method was applied. RESULTS: A total of 45 neonates with HIE received TH during the study period, with 24 patients (53%) in the TH+EPO group. After matching, each group enrolled 13 cases. No significant difference in mortality or hospital stay between the two groups was noted. During the first 3 days, the patients in the TH+EPO group showed significantly higher blood pressure (BP) than those in the TH group ( p < 0.05 on day 1). The TH+EPO group showed trends of higher blood hemoglobin ( p > 0.05) and creatinine ( p > 0.05) levels and lower estimated glomerular filtration rate ( p > 0.05) and urine output ( p > 0.05) during the first 2 weeks than TH group. CONCLUSION: The use of EPO in addition to TH is safe for neonates with HIE. The neonates with moderate or severe HIE who received EPO may have a lesser risk of hypotension than those who received TH alone. Further clinical studies on renal and cardiac functions and long-term neurological effects of EPO are required.

Nephrocalcinosis in children who received high-dose vitamin D.

BACKGROUND: Vitamin D supplements are readily available as over-the-counter preparations. However, although rare, cases of vitamin D overdose still occur and are associated with nephrocalcinosis and life-threatening hypercalcemia. Errors in manufacturing of nutritional supplements may be a cause of vitamin D intoxication in children. This study aimed to identify factors associated with vitamin D overdose-related nephrocalcinosis in children due to manufacturing errors in supplements. METHODS: This retrospective study reviewed medical charts of pediatric patients with non-registered supplement-related vitamin D overdose at a tertiary referral hospital between 2006 and 2011. Clinical and laboratory characteristics of patients with or without nephrocalcinosis were evaluated. Receiver operating characteristics curve and area under the receiver operating characteristics curve were used to determine the most predictive value of each characteristic. RESULTS: A total of 44 patients (males: 29; age: 7-62 months) were included. Age ≤ 16.5 months, body weight ≤ 10.25 kg, body height ≤ 78.5 cm, body surface area (BSA) ≤ 0.475 m2, 25-hydroxyvitamin D3 ≥ 143 ng/mL, and calcium ≥ 10.65 mg/dL were predictive of developing nephrocalcinosis with a sensitivity and specificity of > 60%. Univariant analysis revealed that BSA was the most significant anthropometric prognostic factor (odds ratio: 12.09; 95% confidence interval: 2.61-55.72; P = 0.001). CONCLUSIONS: Children with smaller BSAs were more vulnerable to high-dose vitamin D3-related nephrocalcinosis. Physicians and parents should be aware of the potential adverse effects of vitamin D overdose in children. A higher resolution version of the Graphical abstract is available as Supplementary information.

Ageing attenuates bone healing by mesenchymal stem cells in a microribbon hydrogel with a murine long bone critical-size defect model.

BACKGROUND: Despite the high incidence of fractures and pseudoarthrosis in the aged population, a potential role for the use of mesenchymal stem cells (MSCs) in the treatment of bone defects in elderly patients has not been elucidated. Inflammation and the innate immune system, including macrophages, play crucial roles in the differentiation and activation of MSCs. We have developed lentivirus-transduced interleukin 4 (IL4) over-expressing MSCs (IL4-MSCs) to polarize macrophages to an M2 phenotype to promote bone healing in an established young murine critical size bone defect model. In the current study, we explore the potential of IL4-MSCs in aged mice. METHODS: A 2 mm femoral diaphyseal bone defect was created and fixed with an external fixation device in 15- to 17-month-old male and female BALB/c mice. Microribbon (µRB) scaffolds (Sc) with or without encapsulation of MSCs were implanted in the defect sites. Accordingly, the mice were divided into three treatment groups: Sc-only, Sc + MSCs, and Sc + IL4-MSCs. Mice were euthanized six weeks after the surgery; subsequently, MicroCT (µCT), histochemical and immunohistochemical analyses were performed. RESULTS: µCT analysis revealed that bone formation was markedly enhanced in the IL4-MSC group. Compared with the Sc-only, the amount of new bone increased in the Sc + MSCs and Sc + IL4-MSC groups. However, no bridging of bone was observed in all groups. H&E staining showed fibrous tissue within the defect in all groups. Alkaline phosphatase (ALP) staining was increased in the Sc + IL4-MSC group. The Sc + IL4-MSCs group showed a decrease in the number of M1 macrophages and an increase in the number of M2 macrophages, with a significant increase in the M2/M1 ratio. DISCUSSION: IL4 promotes macrophage polarization to an M2 phenotype, facilitating osteogenesis and vasculogenesis. The addition of IL4-MSCs in the µRB scaffold polarized macrophages to an M2 phenotype and increased bone formation; however, complete bone bridging was not observed in any specimens. These results suggest that IL4-MSCs are insufficient to heal a critical size bone defect in aged mice, as opposed to younger animals. Additional therapeutic strategies are needed in this challenging clinical scenario.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Sex Differences in Mesenchymal Stem Cell Therapy With Gelatin-Based Microribbon Hydrogels in a Murine Long Bone Critical-Size Defect Model.

Mesenchymal stem cell (MSC)-based therapy and novel biomaterials are promising strategies for healing of long bone critical size defects. Interleukin-4 (IL-4) over-expressing MSCs within a gelatin microribbon (µRB) scaffold was previously shown to enhance the bridging of bone within a critical size femoral bone defect in male Balb/c mice. Whether sex differences affect the healing of this bone defect in conjunction with different treatments is unknown. In this study, we generated 2-mm critical-sized femoral diaphyseal bone defects in 10-12-week-old female and male Balb/c mice. Scaffolds without cells and with unmodified MSCs were implanted immediately after the primary surgery that created the bone defect; scaffolds with IL-4 over-expressing MSCs were implanted 3 days after the primary surgery, to avoid the adverse effects of IL-4 on the initial inflammatory phase of fracture healing. Mice were euthanized 6 weeks after the primary surgery and femurs were collected. MicroCT (µCT), histochemical and immunohistochemical analyses were subsequently performed of the defect site. µRB scaffolds with IL-4 over-expressing MSCs enhanced bone healing in both female and male mice. Male mice showed higher measures of bone bridging and increased alkaline phosphatase (ALP) positive areas, total macrophages and M2 macrophages compared with female mice after receiving scaffolds with IL-4 over-expressing MSCs. Female mice showed higher Tartrate-Resistant Acid Phosphatase (TRAP) positive osteoclast numbers compared with male mice. These results demonstrated that sex differences should be considered during the application of MSC-based studies of bone healing.

Mesenchymal Stem Cells and NF-κB Sensing Interleukin-4 Over-Expressing Mesenchymal Stem Cells Are Equally Effective in Mitigating Particle-Associated Chronic Inflammatory Bone Loss in Mice.

Wear particles from total joint arthroplasties (TJAs) induce chronic inflammation, macrophage infiltration and lead to bone loss by promoting bone destruction and inhibiting bone formation. Inhibition of particle-associated chronic inflammation and the associated bone loss is critical to the success and survivorship of TJAs. The purpose of this study is to test the hypothesis that polyethylene particle induced chronic inflammatory bone loss could be suppressed by local injection of NF-κB sensing Interleukin-4 (IL-4) over-expressing MSCs using the murine continuous polyethylene particle infusion model. The animal model was generated with continuous infusion of polyethylene particles into the intramedullary space of the femur for 6 weeks. Cells were locally injected into the intramedullary space 3 weeks after the primary surgery. Femurs were collected 6 weeks after the primary surgery. Micro-computational tomography (µCT), histochemical and immunohistochemical analyses were performed. Particle-infusion resulted in a prolonged pro-inflammatory M1 macrophage dominated phenotype and a decrease of the anti-inflammatory M2 macrophage phenotype, an increase in TRAP positive osteoclasts, and lower alkaline phosphatase staining area and bone mineral density, indicating chronic particle-associated inflammatory bone loss. Local injection of MSCs or NF-κB sensing IL-4 over-expressing MSCs reversed the particle-associated chronic inflammatory bone loss and facilitated bone healing. These results demonstrated that local inflammatory bone loss can be effectively modulated via MSC-based treatments, which could be an efficacious therapeutic strategy for periprosthetic osteolysis.

The Unusual Immune System of the Naked Mole-Rat.

The immune system plays a critical role in host defense to pathogens, tissue homeostasis, cancer development, and several aging-associated chronic inflammatory diseases. The naked mole-rat (Heterocephalus glaber) is a subterranean rodent with both extraordinary longevity and cancer-resistant phenotypes. Unlike the immune system of standard laboratory rodents, that of the naked mole-rat features a higher myeloid-to-lymphoid ratio, lacks natural killer cells, has higher pro-inflammatory cytokine production in macrophages, and exhibits a novel LPS-responsive neutrophil subset that highly expresses several antimicrobials. Given these unusual features, the potential involvement of the naked mole-rat's immune system in their longevity and cancer-resistance remains enigmatic. In this chapter, we summarize the current knowledge of the immune system in the naked mole-rat, including the immune cell repertoire, the primary and secondary lymphoid organs, and the inflammatory responses to the pathogenic stimulation such as bacterial toxins. We compare these findings to published studies of the other subterranean rodents and discuss how the environmental factors in which they have evolved may have influenced their immune function.

Different Effects of Intramedullary Injection of Mesenchymal Stem Cells During the Acute vs. Chronic Inflammatory Phase on Bone Healing in the Murine Continuous Polyethylene Particle Infusion Model.

Chronic inflammation is a common feature in many diseases of different organ systems, including bone. However, there are few interventions to mitigate chronic inflammation and preserve host tissue. Previous in vitro studies demonstrated that preconditioning of mesenchymal stem cells (pMSCs) using lipopolysaccharide and tumor necrosis factor-α polarized macrophages from a pro-inflammatory to an anti-inflammatory phenotype and increased osteogenesis compared to unaltered MSCs. In the current study, we investigated the local injection of MSCs or pMSCs during the acute versus chronic inflammatory phase in a murine model of inflammation of bone: the continuous femoral intramedullary polyethylene particle infusion model. Chronic inflammation due to contaminated polyethylene particles decreased bone mineral density and increased osteoclast-like cells positively stained with leukocyte tartrate resistant acid phosphatase (TRAP) staining, and resulted in a sustained M1 pro-inflammatory macrophage phenotype and a decreased M2 anti-inflammatory phenotype. Local injection of MSCs or pMSCs during the chronic inflammatory phase reversed these findings. Conversely, immediate local injection of pMSCs during the acute inflammatory phase impaired bone healing, probably by mitigating the mandatory acute inflammatory reaction. These results suggest that the timing of interventions to facilitate bone healing by modulating inflammation is critical to the outcome. Interventions to facilitate bone healing by modulating acute inflammation should be prudently applied, as this phase of bone healing is temporally sensitive. Alternatively, local injection of MSCs or pMSCs during the chronic inflammatory phase may be a potential intervention to mitigate the adverse effects of contaminated particles on bone.

Suppression of NF-κB-induced chronic inflammation mitigates inflammatory osteolysis in the murine continuous polyethylene particle infusion model.

Wear particle-associated bone loss (periprosthetic osteolysis) constrains the longevity of total joint arthroplasty (TJA). Wear particles induce a prolonged upregulation of nuclear factor kappa B (NF-κB) signaling in macrophages and osteoclasts. Synthetic double-stranded oligodeoxynucleotides (ODNs) can prevent the binding of NF-κB to the promoter regions of targeted genes and inhibit genetic activation. We tested the hypothesis that polyethylene-particle induced chronic inflammatory bone loss could be suppressed by local delivery of NF-κB decoy ODNs in murine in vivo model. Polyethylene particles were continuously infused into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation, and micro-computational tomography and immunohistochemical analysis were performed. Particle-induced chronic inflammation resulted in lower BMD values, an increase in osteoclastogenesis and nuclear translocation of p65, a prolonged M1 pro-inflammatory macrophage phenotype, and a decrease of M2 anti-inflammatory macrophage phenotype. Delayed timing of local infusion of NF-κB decoy ODN for the last 3 weeks reversed polyethylene-particle associated chronic inflammatory bone loss and facilitated bone healing. This study demonstrated that polyethylene-particle associated chronic inflammatory osteolysis can be effectively modulated via interference with the NF-κB pathway; this minimally invasive intervention could potentially be an efficacious therapeutic strategy for periprosthetic osteolysis after TJA.

Interleukin-4 repairs wear particle induced osteolysis by modulating macrophage polarization and bone turnover.

Periprosthetic osteolysis remains as a major complication of total joint replacement surgery. Modulation of macrophage polarization with interleukin-4 (IL-4) has emerged as an effective means to limit wear particle-induced osteolysis. The aim of this study was to evaluate the efficacy of local IL-4 delivery in treating preexisting particle-induced osteolysis. To this end, recently established 8 week modification of murine continuous femoral intramedullary particle infusion model was utilized. Subcutaneous infusion pumps were used to deliver polyethylene (PE) particles into mouse distal femur for 4 weeks to induce osteolysis. IL-4 was then added to the particle infusion for another 4 weeks. This delayed IL-4 treatment (IL-4 Del) was compared to IL-4 delivered continuously (IL-4 Cont) with PE particles from the beginning and to the infusion of particles alone for 8 weeks. Both IL-4 treatments were highly effective in preventing and repairing preexisting particle-induced bone loss as assessed by µCT. Immunofluorescence indicated a significant reduction in the number of F4/80 + iNOS + M1 macrophages and increase in the number of F4/80 + CD206 + M2 macrophages with both IL-4 treatments. Reduction in the number of tartrate resistant acid phosphatase + osteoclasts and increase in the amount of alkaline phosphatase (ALP) + osteoblasts was also observed with both IL-4 treatments likely explaining the regeneration of bone in these samples. Interesting, slightly more bone formation and ALP + osteoblasts were seen in the IL-4 Del group than in the IL-4 Cont group although these differences were not statistically significant. The study is a proof of principle that osteolytic lesions can be repaired via modulation of macrophage polarization.

The efficacy of core decompression for steroid-associated osteonecrosis of the femoral head in rabbits.

Although core decompression (CD) is often performed in the early stage of osteonecrosis of the femoral head (ONFH), the procedure does not always prevent subsequent deterioration and the effects of CD are not fully clarified. The aim of this study is to evaluate the efficacy of CD for steroid-associated ONFH in rabbits. Twelve male and 12 female New Zealand rabbits were injected intramuscularly 20 mg/kg of methylprednisolone once and were divided into the disease control and CD groups. In the disease control group, rabbits had no treatment and were euthanized at 12 weeks postinjection. In the CD group, rabbits underwent left femoral CD at 4 weeks postinjection and were euthanized 8 weeks postoperatively. The left femurs were collected to perform morphological, biomechanical, and histological analysis. Bone mineral density and bone volume fraction in the femoral head in the CD group were significantly higher than in the disease control group. However, no difference in the mechanical strength was observed between the two groups. Histological analysis showed that alkaline phosphatase and CD31 positive cells significantly increased in the males after CD treatment. The number of empty lacunae in the surrounding trabecular bone was significantly higher in the CD group. The current study indicated that CD improved the morphological properties, but did not improve the mechanical strength in the femoral head at early-stage ONFH. These data suggest the need for additional biological, mechanical strategies, and therapeutic windows to improve the outcome of early-stage steroid-associated ONFH.

Modifying MSC Phenotype to Facilitate Bone Healing: Biological Approaches.

Healing of fractures and bone defects normally follows an orderly series of events including formation of a hematoma and an initial stage of inflammation, development of soft callus, formation of hard callus, and finally the stage of bone remodeling. In cases of severe musculoskeletal injury due to trauma, infection, irradiation and other adverse stimuli, deficient healing may lead to delayed or non-union; this results in a residual bone defect with instability, pain and loss of function. Modern methods of mechanical stabilization and autologous bone grafting are often successful in achieving fracture union and healing of bone defects; however, in some cases, this treatment is unsuccessful because of inadequate biological factors. Specifically, the systemic and local microenvironment may not be conducive to bone healing because of a loss of the progenitor cell population for bone and vascular lineage cells. Autologous bone grafting can provide the necessary scaffold, progenitor and differentiated lineage cells, and biological cues for bone reconstruction, however, autologous bone graft may be limited in quantity or quality. These unfavorable circumstances are magnified in systemic conditions with chronic inflammation, including obesity, diabetes, chronic renal disease, aging and others. Recently, strategies have been devised to both mitigate the necessity for, and complications from, open procedures for harvesting of autologous bone by using minimally invasive aspiration techniques and concentration of iliac crest bone cells, followed by local injection into the defect site. More elaborate strategies (not yet approved by the U.S. Food and Drug Administration-FDA) include isolation and expansion of subpopulations of the harvested cells, preconditioning of these cells or inserting specific genes to modulate or facilitate bone healing. We review the literature pertinent to the subject of modifying autologous harvested cells including MSCs to facilitate bone healing. Although many of these techniques and technologies are still in the preclinical stage and not yet approved for use in humans by the FDA, novel approaches to accelerate bone healing by modifying cells has great potential to mitigate the physical, economic and social burden of non-healing fractures and bone defects.

Increased NF-kB activity in osteoprogenitor-lineage cells impairs the balance of bone versus fat in the marrow of skeletally mature mice.

"Senile osteoporosis" is defined as significant aging-associated bone loss, and is accompanied by increased fat in the bone marrow. The proportion of adipocytes in bone marrow is inversely correlated with bone formation, and is associated with increased risk of fracture. NF-κB is a transcription factor that functions as a master regulator of inflammation and bone remodeling. NF-κB activity increases during aging; furthermore, constitutive activation of NF-κB significantly impairs skeletal development in neonatal mice. However, the effects of NF-κB activation using a skeletally mature animal model have not been examined. In the current study, an osteoprogenitor (OP)-specific, doxycycline-regulated NF-κB activated transgenic mouse model (iNF-κB/OP) was generated to investigate the role of NF-κB in bone remodeling in skeletally mature mice. Reduced osteogenesis in the OP-lineage cells isolated from iNF-κB/OP mice was only observed in the absence of doxycycline in vitro. Bone mineral density in the metaphyseal regions of femurs and tibias was reduced in iNF-κB/OP mice. No significant differences in bone volume fraction and cortical bone thickness were observed. Osmium-stained bone marrow fat was increased in epiphyseal and metaphyseal areas in the tibias of iNF-κB/OP mice. These findings suggest that targeting NF-κB activity as a therapeutic strategy may improve bone healing and prevent aging-associated bone loss in aged patients.

Interleukin-4 overexpressing mesenchymal stem cells within gelatin-based microribbon hydrogels enhance bone healing in a murine long bone critical-size defect model.

Mesenchymal stem cell (MSC)-based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, plays a crucial role in the differentiation and activation of MSCs. The anti-inflammatory cytokine Interleukin-4 (IL-4) converts pro-inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus-transduced IL-4 overexpressing MSCs (IL-4 MSCs) that continuously produce IL-4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL-4 MSCs delivered using a macroporous gelatin-based microribbon (µRB) scaffold for healing of critical-size long bone defects in Mice. IL-4 MSCs within µRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL-4 MSCs within µRBs enhanced bone formation and helped bridge the long bone defect. IL-4 MSCs delivered using macroporous µRB scaffold is potentially a valuable strategy for the treatment of critical-size long bone defects.

Macrophage Effects on Mesenchymal Stem Cell Osteogenesis in a Three-Dimensional In Vitro Bone Model.

As musculoskeletal (MSK) disorders continue to increase globally, there is an increased need for novel, in vitro models to efficiently study human bone physiology in the context of both healthy and diseased conditions. For these models, the inclusion of innate immune cells is critical. Specifically, signaling factors generated from macrophages play key roles in the pathogenesis of many MSK processes and diseases, including fracture, osteoarthritis, infection etc. In this study, we aim to engineer three-dimensional (3D) and macrophage-encapsulated bone tissues in vitro, to model cell behavior, signaling, and other biological activities in vivo, in comparison to current two-dimensional models. We first investigated and optimized 3D culture conditions for macrophages, and then co-cultured macrophages with mesenchymal stem cells (MSCs), which were induced to undergo osteogenic differentiation to examine the effect of macrophage on new bone formation. Seeded within a 3D hydrogel scaffold fabricated from photocrosslinked methacrylated gelatin, macrophages maintained high viability and were polarized toward an M1 or M2 phenotype. In co-cultures of macrophages and human MSCs, MSCs displayed immunomodulatory activities by suppressing M1 and enhancing M2 macrophage phenotypes. Lastly, addition of macrophages, regardless of polarization state, increased MSC osteogenic differentiation, compared with MSCs alone, with proinflammatory M1 macrophages enhancing new bone formation most effectively. In summary, this study illustrates the important roles that macrophage signaling and inflammation play in bone tissue formation.

Probing Pedomorphy and Prolonged Lifespan in Naked Mole-Rats and Dwarf Mice.

Pedomorphy, maintenance of juvenile traits throughout life, is most pronounced in extraordinarily long-lived naked mole-rats. Many of these traits (e.g., slow growth rates, low hormone levels, and delayed sexual maturity) are shared with spontaneously mutated, long-lived dwarf mice. Although some youthful traits likely evolved as adaptations to subterranean habitats (e.g., thermolability), the nature of these intrinsic pedomorphic features may also contribute to their prolonged youthfulness, longevity, and healthspan.