Skin Organoids in Proteostasis Research: Early Insights into Aging

Abstract

The decline of proteostasis is a central hallmark of aging, the earliest manifestations of which have remained difficult to capture in human tissues with conventional model systems. The skin is a continuously renewing and environmentally exposed organ offering a uniquely accessible window into aging biology. Skin organoid technologies allow for long-term culturing of human epidermal and full-thickness skin-like tissues that accurately recapitulate important aspects of cellular heterogeneity, spatial organization, and stem cell dynamics. In this perspective, we discuss how skin organoids are beginning to reveal early proteostasis alterations-encompassing impaired protein folding, reduced proteasomal activity, and autophagy dysfunction-that precede overt structural and functional hallmarks of skin aging, with particular emphasis on underexplored regulators- sebaceous gland and sebocyte-specific proteostasis, autophagy, and inflammaging. We also highlight emerging insights, conceptual challenges, and experimental limitations, and outline future directions for integrating skin organoids with skin-on-a-chip, single-cell proteomics, and stress-reporting approaches to advance proteostasis-targeted interventions in skin aging.

INTRODUCTION

Proteostasis, defined as the maintenance of protein homeostasis, is essential for cellular function and organismal health. It encompasses an integrated network of pathways that regulate protein synthesis, folding, trafficking, and degradation, including molecular chaperones, the ubiquitin–proteasome system (UPS), and autophagy–lysosomal pathways (ALP). A progressive decline in proteostasis capacity is now recognized as a core hallmark of aging, contributing to the accumulation of damaged and misfolded proteins, loss of cellular resilience, and functional deterioration across tissues (Hipp et al., 2019; López-Otín et al., 2023). Importantly, proteostasis collapse is thought to occur early in the aging process, often preceding overt structural or functional decline, yet capturing these early events in human tissues remains a major challenge.

The skin represents a particularly attractive model for studying human aging and proteostasis. As a continuously renewing organ with well-defined stem cell compartments, the skin integrates intrinsic aging processes with chronic exposure to extrinsic stressors such as ultraviolet radiation, pollution, and mechanical injury (Böhm et al., 2025). These features make skin aging both experimentally accessible and biologically informative, while also placing exceptional demands on proteostasis networks to maintain tissue integrity over time. Despite these advantages, existing human aging models have significant limitations. Two-dimensional cell cultures lack tissue architecture and long-term proteostasis dynamics, while animal models often fail to recapitulate human-specific aging trajectories and stress responses (Hossain et al., 2024). Ex vivo human skin explants, although physiologically relevant, are limited by short viability and experimental flexibility (Lebonvallet et al., 2010). As a result, early, cell-type–specific proteostasis alterations during human aging remain poorly understood.

Recent advances in skin organoid technologies offer a conceptual breakthrough in this context. By recapitulating key aspects of three-dimensional organization, stem cell niches, and long-term tissue maintenance, skin organoids provide a human-specific platform to interrogate how proteostasis networks deteriorate during aging. As highlighted by emerging work on proteostasis regulation and stress responses (Hipp et al., 2019), such systems hold promise for uncovering early, mechanistic drivers of aging that have thus far remained experimentally inaccessible. Given the rapid advancement of organoid technologies and their increasing adoption in aging research, skin organoids offer a timely and powerful approach to unravel the molecular underpinnings of proteostasis breakdown, model age-related tissue decline, and evaluate potential therapeutic interventions. This perspective aims to highlight the scientific emergence of skin organoids as a platform for proteostasis studies, discuss their relevance for aging research, and explore future directions for leveraging these models in translational and therapeutic discovery.

PROTEOSTASIS DECLINE IN SKIN AGING

Aging is associated with a progressive decline in the proteostasis network, leading to reduced capacity to maintain protein quality and cellular homeostasis. In the skin, this decline has particularly strong consequences because epidermal renewal, barrier maintenance, and continuous exposure to environmental stressors place exceptionally high demands on protein quality control (Bulteau and Friguet, 2021). As a result, even subtle impairments in proteostasis can have outsized effects on tissue integrity and function. Multiple arms of the proteostasis network are affected with age, including the heat shock response, the ubiquitin–proteasome system, and autophagy–lysosomal pathways (Frankowska et al., 2022; Watanabe et al., 2023). The heat shock response, largely regulated by heat shock factor 1 (HSF1) and molecular chaperones, serves as a first line of defense against proteotoxic stress by promoting proper protein folding and preventing aggregation (Dai, 2018; Kmiecik and Mayer, 2022). With progressing age, both the inducibility and robustness of this response decline. This attenuation leads to reduced chaperone expression and compromises the ability of skin cells to buffer misfolded or damaged o intrinsic metabolic stress and extrinsic insults such as ultraviolet radiation (Zhang et al., 2025).

Parallel defects emerge in protein degradation systems. Proteasomal activity decreases with age, in part due to reduced expression and assembly of proteasome subunits, impairing the clearance of damaged or short-lived proteins. In addition, ALP, which are essential for the turnover of long-lived proteins and organelles, become progressively less efficient (Smith and Carroll, 2025). Defects in autophagosome formation, lysosomal function, and cargo recognition contribute to the accumulation of protein aggregates and dysfunctional organelles in aged cells (Liu et al., 2025; Palmer et al., 2025; Wang et al., 2025b). The age-associated decline in proteostasis in skin cells is depicted in Fig. 1. The consequences of proteostasis failure are highly cell-type specific within the skin. Epidermal stem cells show reduced self-renewal capacity and altered differentiation trajectories. Dermal fibroblasts exhibit impaired extracellular matrix (ECM) maintenance and increased cellular senescence, while melanocytes display heightened sensitivity to oxidative and proteotoxic stress (Miny et al., 2025; Nan et al., 2025; Sun et al., 2025). Collectively, these changes contribute to hallmark features of skin aging, including epidermal thinning, reduced elasticity, pigmentation abnormalities, and delayed wound healing.

Fig. 1.

Age-associated decline in proteostasis in skin cells. Schematic comparison of proteostasis regulation in a youthful skin cell (left) versus an aged skin cell (right). In youthful keratinocytes, cellular stress activates heat shock factor 1 (HSF1), leading to robust chaperone (HSP) expression that promotes correct protein folding. Misfolded proteins are efficiently cleared through coordinated proteasomal degradation (UPS) and autophagy, including effective phagophore formation, autophagosome maturation, and lysosomal degradation, thereby maintaining low levels of protein aggregates and healthy mitochondrial function. In contrast, aged skin cells exhibit reduced HSF1 activity and diminished chaperone expression, resulting in increased protein misfolding and aggregation. Proteasomal and autophagy-lysosome pathway (ALP) become impaired, leading to autophagy blockage, accumulation of aggregates and lipofuscin, elevated reactive oxygen species (ROS), mitochondrial dysfunction, and overall proteostasis collapse.

Despite their significance, early proteostasis defects are difficult to detect in vivo. Compensatory mechanisms often mask subtle declines, and aging-related changes accumulate asynchronously across cells and niches. Moreover, human skin aging unfolds over decades, limiting access to early transitional states. Collectively, these findings position proteostasis decline as an early, coordinated, and cell-type–specific hallmark of skin aging rather than a passive downstream consequence of tissue deterioration. The asynchronous emergence of proteostasis defects across epidermal stem cells, fibroblasts, and melanocytes, together with strong compensatory buffering in vivo, obscures early transitional states in human skin. This gap underscores the need for experimental systems capable of resolving initial proteostasis imbalance before irreversible architectural and functional defects arise.

SKIN RESEARCH MODEL FOR PROTEOSTASIS STUDIES

The skin represents a powerful and accessible model for proteostasis research due to its high cellular turnover, continuous environmental exposure, and reliance on tightly regulated protein quality control systems (Eckhart et al., 2019; Morimoto and Cuervo, 2014; Wang and Dreesen, 2018). Human skin-based platforms, across 2D cultures to ex vivo explants and emerging 3D skin organoids, and provide a translationally relevant framework to interrogate proteostasis regulation and therapeutic modulation. However, experimental models differ markedly in their ability to recapitulate proteostasis across spatial and temporal scales. Conventional 2D keratinocyte or fibroblast monolayer cultures remain widely used because of their high scalability, experimental tractability, and compatibility with high-throughput perturbation assays; however, the absence of tissue architecture, mechanical cues, and multicellular interactions fundamentally limits their capacity to model integrated proteostasis networks that depend on stratification, differentiation gradients, and stromal–epithelial crosstalk. Ex vivo skin explant systems partially address these limitations by preserving native tissue organization, extracellular matrix composition, and cell–cell communication, thereby providing a more physiologically relevant context for studying stress responses and protein quality control. Nevertheless, explants are constrained by rapid loss of viability, typically within days, poor scalability, and limited feasibility for longitudinal monitoring of proteostasis dynamics under chronic or aging-related conditions. In contrast, emerging 3D skin organoid models reconstruct key aspects of human skin architecture, including multilayered epidermal stratification and appendage-like structures such as hair follicle buds, and can be maintained over weeks to months (Ahn et al., 2023; Barua et al., 2021; Piasek et al., 2023). This extended lifespan, combined with increased structural and cellular complexity, enables real-time and longitudinal interrogation of proteostasis pathways—including chaperone responses, proteasomal activity, and autophagic flux—under conditions that more closely approximate in vivo physiology. Collectively, these considerations underscore a progressive trade-off between scalability and physiological relevance across skin models and position 3D skin organoids as a particularly powerful platform (Fig. 2) for studying long-term proteostasis regulation in health and disease. Consistent with the concepts discussed in Section 2, Table 1 summarizes key differences in proteostasis-related pathways between conventional 2D keratinocyte/dermal fibroblast cultures and advanced 3D (skin)organoid models, including spheroids and organoids.

Fig. 2.

Comparative overview of skin research models for proteostasis studies. Schematic comparison of commonly used skin research models highlighting their structural features, experimental advantages, and limitations for investigating proteostasis. The table summarizes key parameters across models, including lifespan, structural complexity, real-time proteostasis dynamics, and scalability, illustrating the trade-offs between experimental control, physiological relevance, and throughput.

Table 1.

Probable relevance of proteostasis markers in 2D and 3D (skin)organoid models

Proteostasis Component	Representative Markers/Assays	2D Skin Models (Monolayer Cultures)	3D Skin Models (Organoids, Reconstructed Skin, Skin-on-Chip)	Biological Relevance	References
Protein Folding Capacity	HSP70, HSP90, BiP/GRP78	Transient stress-induced expression; poor long-term maintenance	Sustained basal and inducible chaperone networks resembling native epidermis	Enhanced folding fidelity and buffering capacity in 3D tissue contexts	(Hipp et al., 2019; Lee et al., 2020)
Unfolded Protein Response (UPR)	PERK–eIF2α–ATF4, IRE1α–XBP1s, ATF6	Chronic ER stress signaling due to substrate stiffness and serum exposure	Adaptive, context-dependent UPR activation and resolution	Improved modeling of ER stress physiology and pathology	(Bhattarai et al., 2021; Hetz and Saxena, 2017)
Proteasome Activity	20S/26S proteasome assays, PSMB5, PSMD11	Elevated basal degradation with limited dynamic range	Regulated degradation responsive to aging and stress stimuli	Reflects physiological protein quality control kinetics	(Debrut et al., 2025; Papendorf et al., 2023)
Autophagy Flux	LC3-II/I ratio, p62/SQSTM1, ATG5/7	Frequently dysregulated or constitutively activated	Functional, inducible autophagy with spatial compartmentalization	Enables modeling of tissue proteostasis maintenance and aging	(Asai et al., 2018; Chen et al., 2025; Ma et al., 2022; Oblong et al., 2020; Sánchez-Martín and Komatsu, 2018)
Protein Aggregation	Ubiquitinated proteins, aggresomes, ProteoStat staining	Early aggregation driven by oxidative and mechanical stress	Progressive aggregation linked to differentiation and aging cues	Distinguishes physiological versus pathological aggregation	(Rogel et al., 2010; Yamazaki et al., 2012)
Redox Homeostasis	NRF2 targets (NQO1, HO-1), ROS, GSH:GSSG ratio	Elevated oxidative burden and chronic stress signaling	Balanced redox control with inducible antioxidant responses	Improved coupling between redox state and proteostasis	(Aiello et al., 2023; Cui et al., 2024; Schäfer and Werner, 2015; Soeur et al., 2017)
Protein Turnover Dynamics	Pulse-chase labeling, SILAC proteomics	Accelerated but dysregulated turnover	Tissue-like kinetics with layer-specific regulation	Faithful modeling of epidermal homeostasis	(Dong et al., 2022; Leung et al., 2025)
Age-Associated Proteostasis Decline	Chaperone loss, autophagy impairment, aggregation burden	Poorly modeled due to culture-induced stress and early senescence	Progressive decline mimicking intrinsic skin aging	Enables mechanistic aging research	(Bulteau and Friguet, 2021; Hipp et al., 2019; Liu et al., 2024; Yamazaki et al., 2012)

This table summarizes general trends reported across representative studies of 2D and 3D (skin)organoid models and integrates current experimental consensus where direct comparative data, particularly for 3D skin organoid systems, remain limited.

SKIN ORGANOIDS: A NEW EXPERIMENTAL PARADIGM

Three-dimensional skin organoid models composed of fibroblasts and keratinocytes have emerged as valuable platforms for investigating the mechanisms underlying skin aging. Skin organoids preserve the spatial organization, cell–cell interactions, and niche signaling required for coordinated protein quality control across multiple lineages. This architecture is particularly relevant for aging research, as proteostasis collapse unfolds in a tissue-context–dependent manner rather than within isolated cell types.

Skin aging phenotypes can be modeled in organoids through prolonged culture, induction of cellular senescence, or derivation from aged donors. These approaches reproduce hallmark characteristics of aged skin, including reduced ECM production, loss of cellularity, and thinning of the epidermal layer (Diekmann et al., 2016). Importantly, these phenotypes emerge alongside gradual deterioration of proteostasis pathways, allowing direct linkage between molecular mechanisms and tissue-level outcomes.

Recent advances in skin organoids encompass a spectrum of models that differ in cellular composition, complexity, and developmental origin. Epidermal-only organoids, derived from keratinocyte stem cells recapitulate stratification, differentiation, and barrier formation, providing a tractable system to study proteostasis dynamics within the epidermal lineage (Kardeh et al., 2025; Li et al., 2025a; Ramovs et al., 2022). However, full-thickness skin organoids that incorporate both epidermal and dermal compartments, offer a distinct mechanistic advantage by enabling investigation of interlayer communication, ECM remodeling, and stromal influences on proteostasis (Baek et al., 2025; Huang et al., 2025a; Smith et al., 2025; Zhang et al., 2024). A study conducted by Shafiee et al. (2024) established physiologically relevant human skin organoids from iPSCs derived from skin fibroblasts and placental CD34⁺ cells that recapitulate key structural and functional features of native skin, including stratified epidermal layers, pigmented hair follicles, sebaceous glands, Merkel cells, and eccrine sweat glands. Their protocol, based on embryoid body induction combined with TGF-β inhibition, FGF activation, and BMP suppression, represents a significant advance in generating complex human skin appendages in vitro and provides a groundbreaking platform for skin development studies, disease modeling, and regenerative medicine applications (Shafiee et al., 2024). However, prolonged culture of skin organoids further mirrors physiological aging processes, notably the induction of p16 expression over time (Dos Santos et al., 2015).

For proteostasis research, skin organoids offer several key advantages. Their capacity for long-term culture allows progressive proteostasis changes to emerge over time, capturing gradual declines that are difficult to observe in short-lived systems. Organoids also preserve human-specific stress responses, avoiding interspecies differences that complicate interpretation of aging mechanisms in animal models (Zwi-Dantsis et al., 2025). Importantly, the three-dimensional organization of organoids supports cell–cell interactions and niche signaling, which are essential for coordinated proteostasis regulation across stem cells, differentiated cells, and supporting populations. Skin organoids have also provided insight into how age-related changes in the tissue microenvironment contribute to skin degeneration. In particular, fragmentation of collagen within the ECM has been identified as a critical driver of aging. Fibroblast proteostasis decline leads to collagen fragmentation and weakened cell–matrix attachment, resulting in altered TGF-β signaling and diminished ECM synthesis (Fisher et al., 2016; Quan et al., 2013), thereby exacerbating tissue fragility. In full-thickness organoids, these stromal changes propagate to the epidermal compartment, where they can induce keratinocyte stress responses, altered differentiation trajectories, and senescence phenotypes. Such interlayer signaling events are not observable in two-dimensional monocultures or epidermal-only systems lacking stromal context.

Skin organoids can further be classified by their cellular source. iPSC-derived skin organoids recapitulate aspects of skin development and allow modeling of genetic diversity, patient-specific mutations, and rejuvenation-associated epigenetic resetting (Yu et al., 2025). In contrast, adult stem cell–derived organoids retain age-associated features and epigenetic memory, making them particularly valuable for studying aging-related proteostasis decline (Zhao et al., 2025). These complementary approaches enable interrogation of both developmental and aging-associated trajectories of protein quality control. Organoids derived from aged donors display clear histological signs of aging (Adamus et al., 2014; Rachidi and Damour, 2014), with the cyclin-dependent kinase inhibitor p16 identified as a key mediator of these alterations, as demonstrated through experimental modulation of p16 expression in organoids generated from both young and aged cells (Adamus et al., 2014). Together, these features establish skin organoids as a mechanistically faithful platform in which the proteostasis pathways outlined in Section 2 can be examined within an integrated tissue context, setting the stage for exploring how early, cell-type–specific proteostasis defects first emerge during aging, as discussed in Section 5.

Compared with traditional human skin explants, organoids provide greater experimental flexibility, genetic manipulability, and longevity (Huang et al., 2025b; Zeng et al., 2025). Unlike reconstructed skin equivalents, which rely on artificial assembly and limited self-organization, organoids more faithfully recapitulate endogenous tissue dynamics. Seminal studies demonstrating the self-organization and functional maturation of human skin organoids (Li et al., 2025a) and recent refinements in stem cell–derived skin models (Garriga-Cerda et al., 2025; Kim et al., 2024b; Wang et al., 2025a) underscore their potential as a next-generation platform to interrogate proteostasis decline during human skin aging. Collectively, skin organoids represent a conceptual shift from static or endpoint-driven skin aging models to dynamic, self-organizing human systems that preserve tissue architecture, cell–cell interactions, and human-specific stress responses. Their experimental longevity, genetic tractability, and capacity to retain age-associated epigenetic features uniquely position skin organoids to interrogate proteostasis decline as a progressive and mechanistically tractable process during human skin aging.

EARLY PROTEOSTASIS INSIGHTS EMERGING FROM SKIN ORGANOIDS

Skin organoids are beginning to yield important early insights into how proteostasis deteriorates during human aging. One recurring observation across organoid systems is the emergence of age-like proteostasis drift during prolonged culture, even in the absence of overt genetic or environmental insults. Extended maintenance of skin organoids is associated with a gradual accumulation of misfolded or damaged proteins, reduced stress resilience, and altered responsiveness to proteotoxic challenges—features reminiscent of early aging phenotypes observed in vivo. Such temporal changes support the concept that proteostasis decline can arise intrinsically from sustained cellular activity and replication, consistent with theoretical frameworks of aging as a progressive loss of protein quality control (Bell and Tower, 2021; Tower, 2015). At the molecular level, skin organoids exhibit altered expression of key proteostasis components, including molecular chaperones, proteasome subunits, and autophagy-related genes (Eckhart et al., 2024; Jie et al., 2025). Reductions in heat shock protein expression and diminished induction of stress-responsive pathways suggest a blunted capacity to buffer proteotoxic stress over time. Concurrently, changes in UPS and ALP point to compromised protein degradation, potentially leading to the accumulation of long-lived or aggregation-prone proteins (Cavallero et al., 2020; Stabell et al., 2023). These alterations parallel age-associated proteostasis defects observed in multiple tissues, reinforcing the relevance of organoids as aging models.

Importantly, skin organoids enable resolution of cell-type–specific vulnerability within a shared tissue context (Bock et al., 2021; Stabell et al., 2023). Epidermal stem and progenitor cells often display heightened sensitivity to proteostasis imbalance, with early stress signatures emerging before comparable changes in differentiated keratinocytes (Blanpain and Fuchs, 2009; Noormohammadi et al., 2018; Tang et al., 2024). In parallel, dermal fibroblasts within full-thickness organoids may exhibit distinct proteostasis trajectories, reflecting differences in metabolic demand and stress exposure (Cavallero et al., 2020; Sutterby et al., 2022). Such heterogeneity underscores the notion that aging does not proceed uniformly across cell types, even within the same organ. Notably, emerging evidence suggests that proteostasis disruption precedes overt differentiation and structural defects in skin organoids (Wang et al., 2023). Perturbations in protein quality control often manifest before detectable changes in stratification, barrier formation, or lineage commitment, positioning proteostasis decline as an early, potentially causal driver of tissue aging. Recent work linking proteostasis regulation to organismal aging trajectories (Ghosh et al., 2025) further supports the idea that skin organoids can capture initial molecular events that set the stage for later functional decline.

Furthermore, age-associated attenuation of autophagic flux in sebocytes undermines the core proteostasis network responsible for protein quality control and organelle turnover, leading to disrupted lipid metabolism and premature cellular senescence (Hou et al., 2022; Liu et al., 2024). However, investigation of sebaceous gland proteostasis has been constrained by reductionist 2D cultures of immortalized sebocytes and short-lived ex vivo gland preparations, which fail to capture long-term proteostasis dynamics at the tissue level. hiPSC–based 3D skin models provide a powerful opportunity to overcome these limitations by enabling sustained, human-relevant interrogation of autophagy–proteostasis coupling in sebocytes. In an advanced skin equivalent developed by Albouy and colleagues, hiPSC-derived sebocytes self-organized into 3D organoids within a reconstructed dermal–epidermal architecture, where they exhibited lipid droplet accumulation and expressed canonical sebaceous markers, including Keratin 7 (KRT7) and Mucin 1 (MUC1). Importantly, the integration of sebocyte organoids into a fibroblast-derived extracellular matrix creates a physiologically relevant niche to study how age-related proteostasis collapse, particularly autophagy dysfunction, drives sebaceous gland decline during skin aging (Albouy et al., 2018). Moreover, a recognized limitation of iPSC-derived organoids as aging models is that cellular reprogramming resets epigenetic age and telomere-associated signatures to a fetal-like state (Horvath et al., 2018; Mertens et al., 2015; Xu et al., 2023), thereby masking intrinsic hallmarks of biological aging. The rejuvenation effect complicates interpretation of proteostasis decline in long-term cultures, as such changes may reflect culture-induced stress rather than aging phenotypes. To address this, strategies including progerin overexpression, chemical induction of senescence such as via DNA damage response activators or mitochondrial stressors, and partial reprogramming approaches that preserve age-associated epigenetic marks while restoring cellular plasticity have been proposed (Hossain et al., 2024; Pennacchi et al., 2015; Weinmüllner et al., 2020). Importantly, advances in lineage reprogramming and organoid engineering further highlight the translational potential of such approaches, as demonstrated by Sun et al., who showed that human epidermal keratinocytes can be reprogrammed into sweat gland–like cells and assembled into functional 3D organoids capable of regenerating sweat glands in vivo (Sun et al., 2021), underscoring the feasibility of personalized regenerative strategies despite challenges related to cellular rejuvenation. In parallel, prolonged culture under maturation-promoting conditions, altered growth factor and metabolic environments, mechanical or electrical stimulation, and in vivo maturation following transplantation, all of which enhance tissue maturity and functional specialization. Moreover, long-term passaging under pro-aging metabolic conditions or exposure to inflammatory cytokine milieus can accelerate aging-like proteostasis dysfunction in vitro (Dos Santos et al., 2015; Weinmüllner et al., 2020). Importantly, organoid systems exhibit greater genomic stability and long-term culture capacity than conventional monolayer systems, enabling chronological aging studies over extended durations. In contrast, adult stem cell–derived organoids retain donor-specific epigenetic memory, telomere characteristics, and accumulated somatic mutations, making them particularly advantageous for modeling intrinsic aging trajectories (Rorteau et al., 2022). Here, we propose that these complementary approaches highlight that combining maturation strategies with accelerated aging paradigms, or integrating adult stem cell–derived and iPSC-derived organoid systems, may provide the most physiologically relevant platforms for studying age-dependent proteostasis dysfunction and tissue aging mechanisms.

In summary, insights from skin organoid models indicate that proteostasis imbalance arises early during aging and precedes detectable defects in tissue organization, differentiation, and barrier integrity. These observations support a causal framework in which impaired protein quality control initiates cellular vulnerability and shapes subsequent tissue-level aging trajectories. By resolving early proteostasis imbalance within a physiologically relevant tissue context, skin organoids provide a critical experimental bridge between molecular aging mechanisms and late-stage skin pathology, addressing a longstanding limitation in human skin aging research.

DISSECTING INTRINSIC AND EXTRINSIC AGING USING SKIN ORGANOIDS

Skin organoids provide a uniquely tractable platform to disentangle intrinsic and extrinsic drivers of human aging under tightly controlled conditions. Intrinsic aging can be modeled through prolonged organoid culture and serial passaging, which recapitulate replicative stress, telomere attrition, and persistent DNA damage responses characteristic of cellular senescence (Gorgoulis et al., 2019; Wang et al., 2024). Within this framework, organoids enable investigation of mitochondrial–proteostasis crosstalk, whereby age-associated mitochondrial dysfunction exacerbates proteostatic collapse and tissue decline, processes that are difficult to resolve in vivo (Martic et al., 2023; Velarde et al., 2015). Although direct longitudinal measurements of proteostasis decline in aging skin organoids remain limited, existing studies in related epithelial organoid systems demonstrate accumulation of oxidatively modified proteins and altered autophagy markers during extended culture, suggesting that organoids can capture early stages of intrinsic proteostatic stress.

In parallel, extrinsic aging mechanisms can be experimentally imposed through defined environmental insults, including ultraviolet radiation, oxidative stress, or chemical exposures. Skin and epithelial organoids subjected to UV irradiation reproduce key features of photoaging, such as proteotoxic stress, ECM remodeling, and impaired regenerative capacity, closely mirroring human extrinsic aging phenotypes (Cavallero et al., 2020; Kim et al., 2024b; Miyake et al., 2019; Olivatti et al., 2020; Sutterby et al., 2022). For example, UV-B irradiation has been reported to increase oxidative protein damage, promote accumulation of autophagy adaptor proteins such as p62, and disrupt proteasome activity in stratified epidermal models, particularly within basal keratinocyte layers. These proteostasis-related alterations are accompanied by increased matrix metalloproteinase expression and reduced collagen deposition, closely mirroring molecular and structural features of human photoaged skin. Importantly, while these studies demonstrate the feasibility of modeling extrinsic aging in organoid-based systems, comprehensive and systematic profiling of proteostasis markers—such as carbonylated proteins, chaperone expression dynamics, proteasome activity, and autophagic flux—remains sparse in skin organoids. This gap highlights a critical opportunity for future research to leverage advanced skin organoid platforms, in combination with single-cell proteomics and spatial analyses, to more precisely map how intrinsic and extrinsic stressors converge on proteostasis decline. Skin organoids may allow these extrinsic insults to propagate through dermal–epidermal crosstalk, revealing how localized damage in one compartment influences proteostasis and aging phenotypes in another. Fig. 3 shows skin organoids as a tractable experimental platform to model both endogenous aging trajectories and environmentally driven stress responses that collectively shape proteostasis dysfunction during skin aging.

Fig. 3.

Intrinsic and extrinsic pathways driving aging and proteostasis decline in skin organoids. Schematic illustration of how healthy skin organoids transition toward an aged or senescent state through converging intrinsic and extrinsic mechanisms. The intrinsic pathway (left) reflects replicative and biological aging processes that emerge during long-term organoid culture, including progressive telomere shortening and cell-cycle arrest mediated by canonical senescence regulators such as p16 and p21. Whereas, the extrinsic pathway (right) represents stress-induced and environmental insults, such as ultraviolet (UV) irradiation or oxidative stress (e.g., H₂O₂ exposure), which trigger DNA damage responses and extracellular matrix (ECM) degradation. Although initiated by distinct cues, both pathways converge on common downstream outcomes characterized by loss of proteostasis, accumulation of misfolded or aggregated proteins, and induction of the senescence-associated secretory phenotype (SASP).

By decoupling genetic background from environmental exposure, organoids offer unparalleled experimental control over dose, timing, and cellular context, allowing direct comparison of intrinsic versus extrinsic aging trajectories. This level of experimental control is difficult to achieve in vivo and unattainable in simpler in vitro systems. As a result, the ability of skin organoids to integrate intrinsic and extrinsic aging cues through proteostasis-centered mechanisms provides a foundation for predictive aging models and targeted interventions

LIMITATIONS OF CURRENT SKIN ORGANOID AGING MODELS

Despite promise, organoid models have some conceptual and technical limitations that must be acknowledged in aging research. Most organoid systems lack key non-epithelial components such as immune cells, vasculature, and innervation (Lee and Koehler, 2021; Lee et al., 2020; Sutterby et al., 2022), all of which play critical roles in tissue homeostasis and age-related decline. The absence of these elements constrains the ability to model complex intercellular interactions that shape proteostasis and inflammatory aging responses. Furthermore, distinguishing intrinsic aging phenotypes from culture-induced stress remains challenging; prolonged in vitro culture can itself elicit metabolic, oxidative, or replicative stress (Sutterby et al., 2022) that may confound interpretation of age-associated changes. While prolonged culture of skin organoids has been associated with proteostasis drift and increased expression of senescence markers such as p16NK4a, it is essential to distinguish authentic biological aging signatures from artifacts induced by in vitro stress conditions. In this context, we propose that nutrient and oxygen gradients within organoid cores, accumulation of metabolic waste, and prolonged exposure to supraphysiological growth factor concentrations can activate stress-response pathways that phenocopy aspects of aging, including oxidative damage, UPR activation, and transient cell cycle arrest. To differentiate these effects, investigators should combine aging-specific biomarkers such as telomere attrition, epigenetic clock acceleration, persistent DNA damage foci (γH2AX/53BP1), altered lamin B1 levels, and decline in proteasome and autophagy flux—with stress-specific readouts including HIF-1α stabilization, ATF4/CHOP induction, NRF2 activation, and mitochondrial membrane depolarization. Importantly, temporal persistence of these signatures following optimization of culture conditions such as improved perfusion, reduced organoid size, or air–liquid interface culture can help distinguish stable aging phenotypes from reversible metabolic stress responses (Park et al., 2024; Zwi-Dantsis et al., 2025). Integrating single-cell transcriptomics and proteomics further enables resolution of cell state heterogeneity and identification of conserved aging trajectories distinct from acute stress programs (Li et al., 2025b), thereby enhancing the interpretability and translational relevance of organoid-based aging studies.

Moreover, methodological variability across differentiation protocols, culture conditions, and handling practices introduces additional noise, making it difficult to compare results across laboratories or to establish universally accepted aging signatures. These inconsistencies are compounded by the lack of standardized, quantitative readouts for proteostasis in organoid systems, inhibiting robust assessment of interventions targeting protein quality control pathways. To advance the field, integration of missing tissue components through co-culture or bioengineering, rigorous controls for culture stressors, harmonization of protocols, and development of validated proteostasis assays are needed. Addressing these limitations will enhance the fidelity of organoids as models of human aging and their utility for translational geroscience.

TOWARD PREDICTIVE HUMAN SKIN AGING MODEL AND PROTEOSTASIS-TARGETED INTERVENTIONS

Emerging technologies are reshaping how human skin aging can be modeled and predicted. The convergence of skin organoid models with high-resolution single-cell proteomics (Elias et al., 2019; Stabell et al., 2023), biological aging clocks, and organ-on-chip platforms offers new opportunities to quantify proteostasis dynamics in a human-relevant context. Single-cell proteomic technologies enable detailed mapping of protein quality control pathways within individual cell types, revealing aging trajectories that are obscured in bulk analyses. While human skin–equivalent (HSE) organoid systems capture key aspects of epidermal differentiation and cellular diversity, they should not be viewed as fully faithful surrogates of in vivo human skin. Single-cell–level analyses conducted by Adam et al; reveal that, alongside conserved differentiation trajectories, HSEs display expanded basal stem cell programs, aberrant terminal differentiation, and non-physiological keratinocyte states driven by culture-dependent signaling. Partial phenotypic rescue following xenograft transplantation underscores the importance of the native microenvironment, while simultaneously revealing new lineage biases imposed by factors such as hypoxia (Stabell et al., 2023). These observations argue that future innovation in skin organoid platforms must move beyond structural mimicry toward dynamic integration of microenvironmental, metabolic, and spatial cues to achieve true physiological relevance. Coupling these data with molecular aging clocks such as epigenetic or proteomic estimators, which have demonstrated robust multi-tissue age prediction and associations with disease risk, provides quantitative benchmarks to gauge biological age in vitro relative to in vivo contexts (Horvath et al., 2018). Such clocks have demonstrated robust age prediction across tissues and strong associations with disease risk. When applied to skin organoids, they offer objective metrics to assess aging trajectories and proteostasis decline under controlled experimental conditions.

Further physiological relevance can be achieved by embedding skin organoids within microfluidic organ-on-chip platforms. These systems enable controlled fluid flow, mechanical forces, and inter-tissue crosstalk, thereby better approximating systemic aging environments. Integrated platforms may function as early-warning systems, detecting subtle shifts in proteostasis or aging clock readouts in response to genetic, environmental, or pharmacological perturbations long before overt phenotypes emerge. This approach aligns with recent efforts to unify multi-scale biomarkers of aging into predictive frameworks (Fernandez et al., 2023; Pusparum et al., 2025; Theodorakis et al., 2024) and suggests a path toward actionable models that forecast individual aging trajectories and therapeutic responses. Advanced skin-on-chip configurations further expand modeling capabilities. Perfusable endothelial channels can capture age-dependent vascular dysfunction and nutrient gradients (Chaturvedi et al., 2022; Middelkamp et al., 2023), while immune-competent chips allow interrogation of inflammaging and immune–proteostasis interactions (Morrison et al., 2024). Emerging evidence indicates that age-associated proteostasis decline is tightly coupled to inflammaging (Jarrold et al., 2022), with immune cells playing central regulatory roles in protein quality control. In particular, macrophages contribute to proteostasis maintenance through the clearance of misfolded proteins, extracellular aggregates, and damaged cellular debris, while cytokines such as IL-6, TNF-α, and IFN-γ modulate autophagy, proteasomal activity, and stress response pathways in resident skin cells (Guimarães et al., 2021; Zou et al., 2025). The absence of immune components in most current skin organoid systems therefore represents a critical limitation for modeling aging-associated proteostasis dysfunction. Recent advances in immunocompetent skin-on-chip and multi-lineage co-culture platforms integrating keratinocytes, fibroblasts, endothelial cells, and immune populations now enable the study of immune–proteostasis crosstalk under controlled inflammatory and aging-like conditions (Bergers et al., 2016; Middelkamp et al., 2023). Very recent, Tan et al. (2026) developed a human-based skin–lymphoreticular organ-on-chip platform that couples reconstructed skin with a self-assembled lymphoid-like unit composed of fibroblastic reticular cells, lymphatic endothelial cells, and CD4⁺ T cells. This system successfully reproduced immune cell motility, lymphoid compartmentalization, and inflammatory skin–immune crosstalk. Upon exposure to atopic-like skin conditions or chemical sensitizers, the model induced robust T cell migration and infiltration, which were reversibly suppressed by anti-inflammatory drugs, demonstrating functional pharmacological responsiveness (Tan et al., 2026). This skin–lymphoid chip represents a significant advancement toward physiologically relevant modeling of inflammatory skin diseases and immunomodulatory drug screening in human-based microphysiological systems. Similarly, incorporating macrophages or myeloid-derived cells into skin organoid and microphysiological systems offers a promising strategy to recapitulate inflammaging-driven proteostasis collapse and to evaluate immunomodulatory interventions alongside classical proteostasis-targeting therapeutics. These next-generation immune-integrated skin models are poised to significantly enhance the physiological relevance and translational potential of organoid-based aging research.

Furthermore, multi-organ chip configurations linking skin organoids with liver or adipose modules further permit assessment of endocrine and metabolic influences on cutaneous aging (Brandauer et al., 2025). When coupled with cyclic mechanical strain or controlled oxidative environments, these platforms provide dynamic contexts in which aging clocks and proteostasis signatures can be longitudinally tracked (Furrer and Handschin, 2025; Shu et al., 2025; Voicu et al., 2025). Fig. 4 highlights skin-on-a-chip technology as a versatile, translational platform bridging mechanistic studies of skin aging with data-driven precision medicine.

Fig. 4.

Integrated skin-on-a-chip platform for multi-omics analysis, aging clock modeling, and drug discovery. Schematic representation of a perfused skin-on-a-chip system that recapitulates key aspects of human skin physiology under controlled microfluidic conditions. The platform supports multi-omics analyses, integration of chip-derived datasets with aging clocks and artificial intelligence (AI)–based approaches, and high-throughput drug screening.

Beyond modelling, skin organoids are emerging as versatile preclinical platforms for evaluating proteostasis-targeted interventions. Age-related loss of protein quality control promotes the accumulation of misfolded proteins and functional decline, and strategies that making proteostasis a promising therapeutic avenues in geroscience and dermatology (Calamini and Morimoto, 2012). For example, small molecules or biologics that activate the proteasome can enhance degradation of damaged proteins and reduce proteotoxic stress (George and Tepe, 2021; West et al., 2012), a mechanism implicated in delayed aging phenotypes in model systems. Likewise, induction of autophagy facilitates lysosomal clearance of aggregated proteins and dysfunctional organelles, with autophagy modulators shown to extend health span in diverse models and offering a tractable target for intervention (Moore, 2020; Rubinsztein et al., 2012; Sun et al., 2016). Consistent with the broader view that autophagy represents a tractable therapeutic target, UVA-induced autophagy in epidermal keratinocytes has been shown to facilitate the clearance of oxidized phospholipids and protein aggregates, thereby contributing to cellular homeostasis under stress conditions (Zhao et al., 2013). These findings support the concept that modulation of autophagic pathways can enhance stress resilience in skin cells, aligning with evidence from diverse model systems in which autophagy activation promotes cellular health and functional maintenance.

Upregulation of molecular chaperones, which assist protein folding and prevent aggregation, represents an additional axis for proteostasis modulation (Babizhayev et al., 2012). Leveraging skin organoids for high-throughput compound screening enables systematic assessment of proteasome activators, autophagy inducers, and chaperone enhancers within a human tissue context that recapitulates epidermal and dermal complexity. Critically, patient-derived organoids can capture individual genetic and environmental aging trajectories, offering a personalized lens on proteostasis dysregulation and drug responsiveness. Such personalized models could inform tailored approaches in dermatology for age-associated conditions like photoaging and impaired wound healing, while also serving as a bridge to broader geroprotective strategies.

Importantly, skin organoids offer distinct advantages over conventional 2D cultures for evaluating therapeutic interventions targeting proteostasis. Unlike monolayer systems, 3D skin organoids recapitulate epidermal stratification, barrier formation, and extracellular matrix architecture, enabling more physiologically relevant assessment of drug penetration across the stratum corneum and diffusion into deeper epidermal and dermal compartments (Abaci et al., 2017; Imran et al., 2024). This is particularly critical for topical or transdermal compounds, where therapeutic efficacy depends on both bioavailability and barrier integrity. Moreover, organoids allow simultaneous evaluation of efficacy and tissue-level toxicity within a multicellular, spatially organized microenvironment (Nguyen and Yoo, 2025), capturing responses such as keratinocyte differentiation defects, stem cell exhaustion, or fibroblast stress that are not detectable in 2D systems. Consequently, like other organoids model, skin organoids may provide a superior platform for screening proteostasis-targeting therapeutics by integrating pharmacodynamics, barrier function, and tissue-specific safety profiles in a single experimentally tractable model (Kim et al., 2024a). By integrating organoid biology with mechanistic insights from proteostasis-targeted drug discovery depicted in Fig. 5 and aging theory, this framework holds translational relevance for both clinical dermatology and the broader goal of extending healthy human lifespan.

Fig. 5.

Therapeutic target map for restoring proteostasis in aged skin cells. Schematic overview illustrating major proteostasis defects in aged keratinocytes and corresponding therapeutic intervention points. Aging-associated accumulation of misfolded protein aggregates, lipofuscin, and reactive oxygen species (ROS) reflects a coordinated decline in chaperone capacity, proteasomal degradation, and autophagy–lysosomal flux. (1) Activation of heat shock factor 1 (HSF1) through chaperone enhancers promotes nuclear translocation and restores transcription of molecular chaperones, thereby improving protein folding capacity. (2) Modulation of the unfolded protein response (UPR) and pharmacological activation of the proteasome enhance clearance of damaged and aggregated proteins. (3) Autophagy inducers stimulate autophagosome formation and lysosomal degradation, facilitating removal of lipofuscin and dysfunctional organelles.

CONCLUSION

Skin organoids are emerging as a transformative experimental system for capturing early, subclinical proteostasis perturbations that precede overt features of human skin aging. By enabling long-term interrogation of human tissue architecture, stem cell dynamics, and stress responses, these models offer a unique opportunity to disentangle intrinsic aging mechanisms from environmentally induced damage. While current limitations including incomplete maturation, lack of systemic inputs, and variability across platforms remain significant, rapid advances in organoid engineering, single-cell and spatial omics, and proteostasis stress-reporting technologies are poised to overcome these barriers. Integrating skin organoids with these complementary approaches will be essential for constructing predictive human aging models and for identifying early, targetable proteostasis vulnerabilities. Such efforts have the potential to redefine how skin aging is studied and to accelerate the development of mechanism-based interventions aimed at preserving tissue function across the lifespan.