MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes.

The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney.

The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an astonishing amount of energy to maintain its functions. In kidney cells, mitochondria produce adenosine triphosphate (ATP) and help maintain kidney function. Due to aging, the efficiency of kidney functions begins to decrease. Dysfunction in mitochondria and cristae, the inner folds of mitochondria, is a hallmark of aging. Therefore, age-related kidney function decline could be due to changes in mitochondrial ultrastructure, increased reactive oxygen species (ROS), and subsequent alterations in metabolism and lipid composition. We sought to understand if there is altered mitochondrial ultrastructure, as marked by 3D morphological changes, across time in tubular kidney cells. Serial block facing-scanning electron microscope (SBF-SEM) and manual segmentation using the Amira software were used to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented, compared to the 3-month, with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we show that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney due to aging. Disruption of the MICOS complex shows altered mitochondrial calcium uptake and calcium retention capacity, as well as generation of oxidative stress. We found significant, detrimental structural changes to aged kidney tubule mitochondria suggesting a potential mechanism underlying why kidney diseases occur more readily with age. We hypothesize that disruption in the MICOS complex further exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, thus impacting kidney health.

Vascularized liver-on-a-chip model to investigate nicotine-induced dysfunction.

The development of physiologically relevant in vitro systems for simulating disease onset and progression and predicting drug metabolism holds tremendous value in reducing drug discovery time and cost. However, many of these platforms lack accuracy in replicating the tissue architecture and multicellular interactions. By leveraging three-dimensional cell culture, biomimetic soft hydrogels, and engineered stimuli, in vitro models have continued to progress. Nonetheless, the incorporation of the microvasculature has been met with many challenges, specifically with the addition of parenchymal cell types. Here, a systematic approach to investigating the initial seeding density of endothelial cells and its effects on interconnected networks was taken and combined with hepatic spheroids to form a liver-on-a-chip model. Leveraging this system, nicotine's effects on microvasculature and hepatic function were investigated. The findings indicated that nicotine led to interrupted adherens junctions, decreased guanosine triphosphate cyclohydrolase 1 expression, impaired angiogenesis, and lowered barrier function, all key factors in endothelial dysfunction. With the combination of the optimized microvascular networks, a vascularized liver-on-a-chip was formed, providing functional xenobiotic metabolism and synthesis of both albumin and urea. This system provides insight into potential hepatotoxicity caused by various drugs and allows for assessing vascular dysfunction in a high throughput manner.

YAP-Driven Malignant Reprogramming of Epithelial Stem Cells at Single Cell Resolution.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ cell tracing approaches with spatiotemporally controlled oncogene activation and tumor suppressor inhibition to unveil the processes underlying oral epithelial progenitor cell reprogramming into cancer stem cells (CSCs) at single cell resolution. This revealed the rapid emergence of a distinct stem-like cell state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal (pEMT) invasive gene programs. Interestingly, CSCs harbor limited cell autonomous invasive capacity, but instead recruit myeloid cells to remodel the basement membrane and ultimately initiate tumor invasion. CSC transcriptional programs are conserved in human carcinomas and associated with poor patient survival. These findings illuminate the process of cancer initiation at single cell resolution, thus identifying candidate targets for early cancer detection and prevention.

'Chip'-ing away at morphogenesis - application of organ-on-chip technologies to study tissue morphogenesis.

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology.

Direct reprogramming of oral epithelial progenitor cells to cancer stem cells at single cell resolution in vivo.

Tumor initiation represents the initial step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Most studies investigating cancer-driving mechanisms in solid tumors rely on analyses of established malignant lesions, and thus cannot directly capture processes underlying the reprogramming of normal progenitor cells into cancer cells. Here, using spatiotemporally controlled oncogene expression in a genetically engineered system we demonstrate that concomitant YAP activation and HPV E6-E7 -mediated inhibition of tumor suppressive pathways is sufficient to rapidly reprogram oral epithelial progenitor cells (OEPCs) into cancer stem cells (CSCs). Single cell analyses of these nascent CSCs revealed hallmark transcriptional programs driving tumor initiation. Importantly, these CSC-enriched expression signatures distinguish normal tissue from malignant head and neck tumors and are associated with poor patient survival. Elucidating mechanisms underlying OEPC to CSC reprogramming may offer new insights to halt the conversion of premalignant cells into invasive carcinoma. HIGHLIGHTS: YAP and HPV E6-E7 reprogram oral epithelial progenitor cells into cancer stem cells. Single cell analyses reveal the transcriptional architecture of tumor initiation.CSC transcriptional programs distinguish normal tissue from carcinoma.CSC signatures are associated with poor head and neck cancer survival.

Juneteenth in STEMM and the barriers to equitable science.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.

Strategies for Regenerative Vascular Tissue Engineering.

Vascularization remains one of the key challenges in creating functional tissue-engineered constructs for therapeutic applications. This review aims to provide a developmental lens on the necessity of blood vessels in defining tissue function while exploring stem cells as a suitable source for vascular tissue engineering applications. The intersections of stem cell biology, material science, and engineering are explored as potential solutions for directing vascular assembly.

Directing Cholangiocyte Morphogenesis in Natural Biomaterial Scaffolds.

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch-dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue.

Racial discrimination and other adverse childhood experiences as risk factors for internalizing mental health concerns among Black youth.

Adverse childhood experiences (ACEs) have been consistently linked to a reduction in healthy psychological adjustment among youth. Emergent evidence suggests that there are culturally specific ACEs, such as racial discrimination, that are particularly harmful to the mental health of Black youth. However, the psychological impact of racial discrimination on the mental health of Black youth relative to other ACEs remains underexplored. The present study aimed to address this gap by examining the extent to which racial discrimination was associated with other ACEs and elucidating the unique associations between children's experiences of racial discrimination and internalizing problems (i.e., depression, anxiety), after controlling for other ACEs. Data consisted of a subsample of Black children from the National Survey of Children's Health (N = 8,672; Mage  = 9.8 years; 51.1% male). Bivariate analyses illustrated that racial discrimination was positively associated with the co-occurrence of all other ACEs measured within the current study. Multivariable analyses using generalized linear mixed models revealed that racial discrimination was significantly associated with youth diagnoses of depression, adjusted odds ratio (aOR) = 1.35, 95% CI [1.23, 1.49], and anxiety, aOR = 1.39, 95% CI [1.31, 1.47], after controlling for other ACEs and sociodemographic covariates. The findings demonstrate that racial discrimination is comparably associated with youth internalizing problems relative to ACEs conventionally examined within the childhood trauma literature. The importance of these results, including how this knowledge can be leveraged to inform clinical practice and policy to promote the positive mental health of Black youth, are also discussed.

Can't touch this: Stromal-mediated ductal proliferation.

Unraveling and replicating how cells communicate to regenerate organs remains one of the most compelling biological problems of our time. In this issue of Cell Stem Cell, Cordero-Espinoza et. al (2021) untangle how a subpopulation of liver mesenchymal cells residing adjacent to the bile ducts regulate biliary cell proliferation.

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

Collagen fiber structure guides 3D motility of cytotoxic T lymphocytes.

Lymphocyte motility is governed by a complex array of mechanisms, and highly dependent on external microenvironmental cues. Tertiary lymphoid sites in particular have unique physical structure such as collagen fiber alignment, due to matrix deposition and remodeling. Three dimensional studies of human lymphocytes in such environments are lacking. We hypothesized that aligned collagenous environment modulates CD8+ T cells motility. We encapsulated activated CD8+ T cells in collagen hydrogels of distinct fiber alignment, a characteristic of tumor microenvironments. We found that human CD8+ T cells move faster and more persistently in aligned collagen fibers compared with nonaligned collagen fibers. Moreover, CD8+ T cells move along the axis of collagen alignment. We showed that myosin light chain kinase (MLCK) inhibition could nullify the effect of aligned collagen on CD8+ T cell motility patterns by decreasing T cell turning in unaligned collagen fiber gels. Finally, as an example of a tertiary lymphoid site, we found that xenograft prostate tumors exhibit highly aligned collagen fibers. We observed CD8+ T cells alongside aligned collagen fibers, and found that they are mostly concentrated in the periphery of tumors. Overall, using an in vitro controlled hydrogel system, we show that collagen fiber organization modulates CD8+ T cells movement via MLCK activation thus providing basis for future studies into relevant therapeutics.

Genetic, Inflammatory, and Epithelial Cell Differentiation Factors Control Expression of Human Calpain-14.

Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14 Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ∼10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.

Cytoskeletal tension regulates mesodermal spatial organization and subsequent vascular fate.

Morphogenesis during human development relies on the interplay between physiochemical cues that are mediated in part by cellular density and cytoskeletal tension. Here, we interrogated these factors on vascular lineage specification during human-induced pluripotent stem-cell (hiPSC) fate decision. We found that independent of chemical cues, spatially presented physical cues induce the self-organization of Brachyury-positive mesodermal cells, in a RhoA/Rho-associated kinase (ROCK)-dependent manner. Using unbiased support vector machine (SVM) learning, we found that density alone is sufficient to predict mesodermal fate. Furthermore, the long-withstanding presentation of spatial confinement during hiPSC differentiation led to an organized vascular tissue, reminiscent of native blood vessels, a process dependent on cell density as found by SVM analysis. Collectively, these results show how tension and density relate to vascular identity mirroring early morphogenesis. We propose that such a system can be applied to study other aspects of the stem-cell niche and its role in embryonic patterning.

Differential HDAC6 Activity Modulates Ciliogenesis and Subsequent Mechanosensing of Endothelial Cells Derived from Pluripotent Stem Cells.

The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.

Compliant substratum guides endothelial commitment from human pluripotent stem cells.

The role of mechanical regulation in driving human induced pluripotent stem cell (hiPSC) differentiation has been minimally explored. Although endothelial cell (EC) fate from hiPSCs has been demonstrated using small molecules to drive mesoderm induction, the effects of substrate stiffness with regard to EC differentiation efficiency have yet to be elucidated. We hypothesized that substrate compliance can modulate mesoderm differentiation kinetics from hiPSCs and affect downstream EC commitment. To this end, we used polydimethylsiloxane (PDMS)-a transparent, biocompatible elastomeric material-as a substrate to study EC commitment of hiPSCs using a stepwise differentiation scheme. Using physiologically stiff (1.7 MPa) and soft (3 kPa) PDMS substrates, compared to polystyrene plates (3 GPa), we demonstrate that mechanical priming during mesoderm induction activates the Yes-associated protein and drives Wnt/ß-catenin signaling. When mesoderm differentiation was induced on compliant PDMS substrates in both serum and serum-free E6 medium, mesodermal genetic signatures (T, KDR, MESP-1, GATA-2, and SNAIL-1) were enhanced. Furthermore, examination of EC fate following stiffness priming revealed that compliant substrates robustly improve EC commitment through VECad, CD31, vWF, and eNOS marker expression. Overall, we show that substrate compliance guides EC fate by enhancing mesoderm induction through Wnt activation without the addition of small molecules. These findings are the first to show that the mechanical context of the differentiation niche can be as potent as chemical cues in driving EC identity from hiPSCs.