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Aging research has shifted from studying phenotypes to studying in-depth mechanisms in recent decades. However, extrapolating cellular and molecular bases of aging from studying traditional model systems to humans has been challenging. The advent of organoids holds promise for overcoming the limitations of monolayer cell culture and bridging the gap between animal models and humans. Here, we mainly discuss recent paradigms for using organoid models in studying organ aging. Pluripotent stem cells– derived organoid provides a promising platform for simulating the pathophysiology of several aging-related diseases, especially neurodegenerative diseases, and adult stem cells organoids derived from different age groups have been applied to detect aging- related functional changes. We also assess the value of organoid model systems in understanding human aging and aging-related diseases, and identify challenges to be addressed in the future, such as the immaturity of organoids, and effective methods of inducing aging. Keywords: aging, disease modeling, organoid, senescence, stem cell and guide the development of candidate drugs for delaying Introduction human aging. These model systems include animals, such as The elderly population has continued to expand in recent years, nematodes, rodents, non-human primates, and cells of human and without the accompanying healthspan growth, it has led to [3–8] origins.  economic and social burdens. Aging is the major risk factor for  Human aging is a complex physiological process. most chronic diseases, such as cancer, diabetes, cardiovascular Delineating the etiology and pathophysiology of aging, there- disease, dementia, arthritis, sarcopenia, and renal dysfunction, fore, requires experimental models that can recapitulate multi-  that seriously affect the well-being of the elderly. Therefore, faceted pathophysiology and clinical manifestations of human studying the underlying mechanisms of human aging and devel-  aging. However, it is worth noticing that the maximal lifes- oping interventions targeting the aging process have become pan varies greatly across species and different species have the priorities for research worldwide. Considering the biologi- evolved different molecular mechanisms to control their lifes- cal relevance and practicality, various model systems have been [10,11] pans. Many biological processes are very different between selected to investigate the cellular and molecular mechanisms  the human and other organisms, such as brain development, underlying aging, identify potential aging intervention targets,  [14,15] immune response, and drug metabolism. For instance, most laboratory animals have higher rates of pharmacokinet- [16–18] ics than humans. Cells, regardless of established cell lines XS, FS, and YZ contributed equally to the writing of the article. or recently isolated primary cells, are traditionally cultured in CAS Key Laboratory of Genomic and Precision Medicine, Beijing Institute vitro as two-dimensional (2D) monolayers with a limited capac- of Genomics, Chinese Academy of Sciences, China National Center for  ity for physiologic representation. For example, extracellular Bioinformation, Beijing, China, Department of Cell Biology, Duke University d deposition of amyloidogenic peptides, a typical characteristic Medical Center, Durham, NC, USA, State Key Laboratory of Stem Cell of Alzheimer’s disease (AD), cannot be recapitulated in induced and Reproductive Biology, Institute of Zoology, Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing Institute for Stem Cell and pluripotent stem cells (iPSCs)–derived neurons grown as mono- Regenerative Medicine, State Key Laboratory of Membrane Biology, Institute of  layer. Thus, traditional animal and cell culture models cannot Zoology, Chinese Academy of Sciences, Advanced Innovation Center for Human fully reflect the aging-associated physiological and pathologi- Brain Protection, and National Clinical Research Center for Geriatric Disorders, cal states of humans. These limitations of classical models have Xuanwu Hospital Capital Medical University, Beijing, China hindered the translation of basic research findings into clinical *Corresponding author: Weiqi Zhang, CAS Key Laboratory of Genomic and applications. Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China. E-mail: email@example.com; and Guang-Hui Liu, State Compared with the models mentioned above, the recently Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of developed organoid system has been recognized for its great Sciences, Beijing 100101, China. E-mail: firstname.lastname@example.org potential in translational medicine. Organoids are referred to Copyright © 2023 The Chinese Medical Association, Published by Wolters as “Preclinical Models of Human Disease” because they largely Kluwer Health, Inc. This is an open-access article distributed under the terms of recapitulate the physiological cellular compositions and preserve the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 [19,21] human regulatory pathways. Organoids address many lim- (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially itations of cells grown in the traditional 2D culture and bridge without permission from the journal.  the gap between animal models and human beings. For Journal of Bio-X Research (2023) 6:3–14 instance, organoids have higher physiological complexity com- Received: 4 September 2022; Accepted: 15 February 2023 pared to 2D cell culture, are more accessible compared to ani-  http://dx.doi.org/10.1097/JBR.0000000000000139 mals, and better simulate human physiology. Therefore, to a 3 Review AR ticle Journal of Bio-X Research certain extent, the organoid system has the potential to become with self-organizing crypt-villus structures from a single mouse  a more reliable aging research model (Fig. 1). Lgr5-expressing intestinal stem cells in vitro. This was the An organoid is “a collection of organ-specific cell types first reported three-dimensional (3D) organoid culture from derived from stem cells or organ progenitors, self-organized a single ASC, which laid the foundation for many subsequent through cell sorting and spatially restricted lineage commitment organoids works. These include the generation of ASC- or PSC-    like in vivo.” Organoids can be generated from either PSCs, derived neuroectoderm (eg, brain and retina ) and mesen- [30,31]  [33–36] [37–41] including embryonic stem cells (ESCs) and iPSCs, or adult stem doderm (eg, heart, stomach, intestine, liver, [42–46] [47–49] [50–53] cells (ASCs) and are called PSC-derived organoids and ASC- pancreas, lung, and kidney ). Established on top  [54–56] derived organoids, respectively. PSC-derived organoids are of the stem cell technology, the reprogramming technol- [57–60] generated by stepwise differentiation protocols, which recapit- ogy, and the existing knowledge of organogenesis, organoid ulate the signals during gastrulation and organogenesis with technologies continue to develop by integrating new technolo- [25,51,61–63] various growth factors or inhibitors to initiate the development gies. Researchers can now mimic the 3D architecture, of different germ layers (endoderm, mesoderm, and ectoderm). the cell-type composition, and the function of a wide range of On the other hand, ASC-derived organoids are generated by tissues and organs with the organoid culture. The organoid sys- culturing ASCs in the presence of niche factors, a cocktail of tem has emerged as a powerful and pivotal research platform growth factors that maintain ASCs in an undifferentiated state for both developmental biology research and clinical applica- [22,24] while permitting differentiation. Although most organoids tions, such as disease modeling, drug discovery, biobanking, pre-  can be derived from both PSCs and ASCs, certain organoids, cision medicine, and regenerative medicine. [22,25,26] like the brain ones, can only be generated from PSCs. In The history of organoid research has been well described by [24,64] 2009, Hans Clevers’s group generated an intestinal organoid others (Fig. 2). In this review, we focus on discussing recent Figure 1. Comparison of organoids with other model systems. We compare the advantages and disadvantages of the 2D cell culture, Mus musculus, non-hu- man primates, and human organoids used in biological research. Compared with other models, human organoids are highly anticipated for their ability to more realistically mimic the physiological and pathological features of the human body. Different tags are used to represent different relative scores, and they are best suitable (dark green tick), partly suitable (yellow circle), not suitable (red cross). Unpublished data. Figure 2. The history of organoid research. Timeline of milestones for the organoid methodologies. hESCs=human embryonic stem cells, PSC=pluripotent stem cells. 4 Journal of Bio-X Research Review AR ticle implementations of organoid technologies in aging studies. We generated by stepwise differentiation protocols usually resemble highlight the value of organoids as model systems in both mech- fetal-stage tissues and are mainly used to study organogenesis and anistic and translational research for human aging and aging-re- early developmental events, organoids generated from PSCs car- lated disease and point out key challenges that remain to be rying aging-related genetic variations exhibit accelerated emer- addressed. gence of aging-related pathophysiology (Fig. 3A). For instance, many brain organoids of NDDs well reproduce the pathologi- cal phenotypes of these diseases, such as amyloid aggregation, Database retrieval strategy hyperphosphorylated tau protein, and endosome abnormalities  Literature review was electronically performed using PubMed in familial AD. iPSC-derived organoids have higher maturity, database. The following combinations of key words were used more complex culture environment, and intercellular interaction, to initially select the articles: aging model; organoid; organoid which thus can reproduce more complex and diverse pathological and aging; brain organoid; ASCs and aging. Most of the selected features of degenerative diseases. For example, in AD iPSC-de-  articles (90% of all references) were published from 2013 to rived organoids, amyloid β (Aβ) plaques occur spontaneously. 2023. The articles included in this review were selected based on However, iPSC-derived neurons from AD patients without exog- their relevance to the topic. enous induction did not produce Aβ plaques, possibly due to the diffusion of secreted Aβ into the medium due to 2D culture  conditions, reflecting a defect in the 2D cell model. The estab- Applications of organoids in aging research lishment of iPSC-derived organoids thus provides a great oppor- The combination of iPSCs possessing disease-related mutations tunity to recapitulate the progression of aging-related diseases in with organoid technologies provides a promising platform for sim- vitro, allowing researchers to explore the early events of diseases  ulating the pathophysiology of several aging-related diseases, and discover early disease-associated molecular signatures and [66,67] [68–70] [78,79] including neurodegenerative diseases (NDDs), arthritis, markers that can be ideal targets for therapies. Moreover, [71–73] [74,75] diabetes, and cancer. Although PSC-derived organoids drug screenings using organoid models have provided us with Figure 3. Application of organoids in aging research. (A) PSC-derived organoids are potent tools in modeling aging-related diseases, such as neurodegener- ation. (B) ASC-derived organoids reflect the homeostatic or regenerative conditions of their tissue of origin. Identifying and isolating ASCs from young and old donors to generate ASC-derived organoids in vitro will improve our understanding of the molecular mechanism underlying stem cell exhaustion and guide the identification of novel targets for intervening stem cell aging. (C) Other organoids cultured in vitro, especially those established with microfluidic systems, well simulate the aging processes caused by inflammation and external stress. Moreover, the rapid development of organ-on-chip will greatly promote the study of multi-organ pathologies and systemic aging. ASC=adult stem cell, PSC=pluripotent stem cells. Unpublished data. 5 Review AR ticle Journal of Bio-X Research much new information, especially for diseases with complicated provides models for elucidating the interactions between brain  pathogenesis. For instance, integrating mathematical modeling regions. A recent study demonstrates that the cerebral-cor- and human brain organoids from iPSCs seems to be a more effec- tex organoid and the spinal cord organoid can also fuse with tive way to identify potential and optimal drug targets for each the skeletal muscle-organoid to simulate the cortico-motor   risk factor of AD. circuit. Identifying and isolating ASCs from young and old donors In the past decade, researchers have introduced multiple to generate organoids in vitro is feasible and has gradually approaches from diverse aspects to optimize the culture condi- become a common approach to studying aging (Fig. 3B). Stem tion for brain organoids. For example, forming embryoid bod- cell exhaustion is considered the ultimate culprit for cellular ies from single-cell suspensions can reduce variabilities between  [110,111] and organismal aging. Functional attrition of ASCs during batches and slice cultures can minimize internal necrosis  aging reduces tissue repair and regeneration potentials sys- caused by hypoxia and nutrient deficiencies. The advances in [82,83] temically, one of the manifestations of degenerative aging. 3D-printed mini-bioreactors can further increase the through-  Accordingly, cells from aged mice and humans have been put of organoid production. [84–87] reported to have reduced organoid formation efficiency. The brain organoids used for modeling NDDs are generated [20,66] Although it is still unclear whether organoids generated from primarily using iPSCs derived from patients, and only a ASC isolated from old donors manifest hallmarks of aging in proportion of them are derived from genetically modified ESCs. the absence of the aging niche, several aging-related markers can Brain organoids containing genetic variations associated with be reproduced in such organoids. Thus, organoids derived from NDDs have become a promising model for studying the early aged individuals are potent models for studying the molecular pathological features of these diseases (Fig. 4). Findings from mechanisms underlying stem cell aging. these studies will support the development of clinical diagnoses Some tissues and organs, such as skin and joints, cannot be and interventions. mimicked in vitro with PSC- or ASC-derived organoids. One of the standard methods to resolve this problem is co-culturing Modeling AD with brain organoids  multiple cell types on scaffolds (Fig. 3C). Moreover, aging is Both whole-brain and forebrain organoids can be used to model a systemic process accompanied by the degeneration of various AD. Brain organoids derived from iPSCs generated from AD tissues and organs. The pathophysiology of some aging-related patients spontaneously develop disease-associated pathologies, diseases is also highly relevant to inter-organ crosstalk. The such as amyloid aggregation, hyperphosphorylation of Tau organ-on-a-chip technology uses microfabricated cell culture proteins (pTau), endosome abnormalities, and cellular apopto- devices that combine biomaterial technologies, microfluidic [113,114] sis (Fig. 4), that can hardly be reproduced in 2D culture systems, and tissue engineering and can simulate the crosstalk   models. Besides, the apolipoprotein E gene (APOE)-mutated between different tissues and organs. Despite its current lim-  brain organoids also display AD-associated phenotypes, such as itations, the organ-on-chip technology has been employed increased α-synuclein, impaired synaptic functions, decreased to study several aging-related diseases, such as rheumatoid     glucocerebrosidase levels, and accumulated lipid droplets. arthritis, hypertensive nephropathy, diabetes, and neu-  Remarkably, exogenous APOE2/3, but not APOE4, partially romuscular diseases (Fig. 3C). With the advances of new rescues these phenotypes, suggesting the positive effects of technologies, the organ-on-chip technology will likely become APOE2/3 in preventing AD. The deletion of the mitochondrial a powerful tool for studying systemic milieus that regulate the enzyme pitrilysin metallopeptidase 1 gene (PITRM1) in brain aging process. organoids also spontaneously leads to the development of AD Below, we will describe the current applications of organoids pathological features, suggesting a link between mitochondrial in aging research in detail.  dysfunctions and AD. With the extension of the culture period, brain organoids can display aging-related pathologi- cal features. The intervention with β and γ secretase inhibitors Pluripotent stem cell–derived organoids reduces Aβ production first and then the tau hyperphosphory- Brain organoids  lation in these brain organoids. Exposing brain organoids to Human brain organoids are derived from PSCs, like ESCs or human serum, a culture condition mimicking the blood-brain iPSCs. Previous in vitro human brain models had established barrier leakage in AD patients, leads to synaptic loss and neural  approaches for stimulating self-organization and inducing dif- network impairments in these brain organoids. Serum acti- [94–99] ferentiation of PSCs. Building upon these works, in 2013, vates glycogen synthase kinase (GSK) pathways and elevates Aβ  Lancaster et al first established a human PSC-derived whole- and pTau levels in human brain organoids, suggesting that GSK brain organoid employing Matrigel to support the 3D organi- is a potential target for AD interventions. zation of the organoid without using patterning growth factors. Impairment in astrocytes has been implicated in various Identities of various brain regions, including the forebrain, the NDDs, especially in AD. However, astrocytes can hardly be dif- [100,118] midbrain, the hindbrain, and the retinal tissues, were recog- ferentiated into brain organoids, limiting the application  nized in this whole-brain organoid. To generate dorsal forebrain of this approach in NDD modeling. Recently, a chimeric   organoids and ventral forebrain organoids, the Pasca brain organoid system has been developed to accelerate the pro- group exposed embryoid bodies to neural induction and dif- duction of functional astrocytes. By generating chimeric brain ferentiation factors. Following that is the generation of various organoids containing neurons and astrocytes with different  region-specific organoids, including the midbrain organoid, APOE allelotypes (APOE3 or APOE4), chimeric cerebral organ-   the cerebellum organoid, the striatal organoid, the cho- oids carrying APOE4 are found to show elevated Aβ and p-Tau   roid plexus organoid, the spinal cord organoid, and the levels, increased lipid droplet formation, and increased accumu-  hypothalamic arcuate organoid. Excitingly, different brain lation of cholesterol in neurons. This chimeric organoid system region-specific organoids can fuse to form assemblies, which provides a unique opportunity to dissect cell-type–specific roles 6 Journal of Bio-X Research Review AR ticle Figure 4. Brain organoids modeling NDDs. iPSCs-derived whole-brain organoid or region-specific brain organoid can well recapitulate the key pathological characteristics of many NDDs, such as AD, PD, A-T, and HD. Brain organoid models facilitate the elucidation of the molecular features of NDDs at the early stage of disease progression and the subsequent development of early intervention drugs. AD=Alzheimer’s disease, APOE4=apolipoprotein E4, APP=amyloid precursor protein, A-T=ataxia-telangiectasia, ATM=ataxia telangiectasia-mutated gene, Aβ=amyloid β-protein, cGAS-STING=the cyclic GMP-AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway, FTD=frontotemporal dementia, HD=Huntington disease, HTT=huntingtin, iPSC=induced pluripotent stem cells, LRRK2-G2019S=leucine-rich repeat kinase 2-G2019S, NDDs=neurodegenerative diseases, PARK7=Parkinson disease 7, PD=Parkinson disease, PITRM1=pitrilysin metallopeptidase 1, PSEN1=Presenilin 1. Unpublished data.  in AD pathogenesis. In order to delve into the mechanism of Modeling Parkinson’s disease with brain organoids tau pathology in the whole-brain organoids of microtubule-as- Parkinson’s disease (PD) is a neurodegenerative disorder that sociated protein tau (MAPT) mutations, mature brain organ- affects mainly the dopaminergic neurons in the midbrain, oids can be digested into individual neurons. Morphological and midbrain dopaminergic neurons (mDANs). Organoids and functional abnormalities of axons as well as mislocalization that recapitulate hallmarks of PD, such as the degradation of and reduced phosphorylation levels of the mutant tau proteins mDANs and the aggregation of α-synuclein, can be generated  in the neurons can be found. Moreover, the electrophysiol- from either iPSCs derived from PD patients or genetically mod- [123–125] ogy platform for brain organoids has been established. With this ified human ESCs (Fig. 4). Several studies have unveiled platform, AD organoids are found to abnormally increase the neurodevelopmental defects in PD organoid models, such as frequency of spontaneous action potentials (Fig. 4). Inhibitor impaired WNT-LMX1A autoregulatory pathway and upregu- [123,124] screens show that the NMDA-type glutamate receptors lated neural progenitor cell marker FOXA2 in mDANs. (NMDAR) antagonist NitroSynapsin can rescue this abnormal Mechanistic studies have revealed molecular targets for delay-  neural activity. The organoid-based high-throughput screen- ing the progression of PD in various mutant backgrounds. ing system holds promises for identifying and validating new The WNT-LMX1A signals are impaired in organoids carrying  drugs for AD treatments. DNAJC6 mutations, which result in the production of mDANs 7 Review AR ticle Journal of Bio-X Research with reduced expressions of midbrain markers and shrunken cell and regulates stem cell functions. It consists of niche cells, cellu-  [135,136] bodies. In LRRK2-G2019S mutant organoids, the expres- lar matrix, and secreted soluble factors. Compared with 2D sion of TXNIP encoding a thiol-oxidoreductase is reported to cell cultures, assessing the proliferation and differentiation abil- be significantly induced. Knocking down TXNIP significantly ity of ASCs in 3D organoid cultures is more viable, as organoids  reduces the aggregation of α-synuclein. In PARK7-mutant partly replicate the complex cellular interactions in tissues and the  PD organoids, a defect in the U1-dependent splicing of the molecular environments that stem cells are exposed to in vivo. PARK7 transcript is identified, which causes the malfunction ASCs from old donors have been used to generate organ-  of mitochondria and the loss of dopaminergic neurons. With oids for aging research (Fig. 5). For example, intestinal epithe- the ability to recapitulate early pathological features of PD, the lial organoids have been established with stem cells from mice midbrain organoid provides a powerful platform for identifying at various stages of life, and those generated from aged mice effective preventive strategies for PD. For example, combinato- accumulate the senescent marker SA-β-gal and increase expres-  rial treatment with small molecules that promote exon inclusion sions of Cdkn1a (p21) and Cdkn2a (p16). To evaluate the and protein expression can restore the dopaminergic neuron use of human organoids for aging studies, DNA methylation loss in PARK7-mutant midbrain organoids. In addition, the rates of organoids derived from human intestines were assessed neural progenitor cells isolated from midbrain organoids can and found to maintain the regional-specific and age-associated  serve as stem cell repertoire for generating functional mDANs epigenetic signatures of their tissues of origin. Similarly, ex  for PD treatment. vivo long-term-cultured mouse colon-derived organoids also mimic epigenetic features of in vivo aging, especially global methylation changes including hypermethylation at promoters Modeling other NDDs with brain organoids and hypomethylation in intronic and intergenic regions. The aging-like spontaneous DNA hypermethylation at promoters With the use of patient iPSCs-derived organoids, NDDs research V600E  promotes Braf -induced transformation. Age-associated has rapidly progressed. Using the state-of-art single-cell transcrip- increase in non-pituitary growth hormone (npGH) and p16 lev- tomic approach in combination with biological assays, research- els and decrease in telomere length are observed in intestinal ers analyzed the cerebral organoid slice model for amyotrophic  organoids cultured for up to 4 months. Moreover, investiga- lateral sclerosis overlapping with frontotemporal dementia. They tions of age-induced changes in niche-stem cell communications identified DNA damage in both astrocytes and neurons and the  reveal that the induction of notum in aged Paneth cells, the niche potential of GSK2606414 in rescuing this phenotype. In a cells supporting the intestinal stem cells, inhibits WNT signaling brain organoid model for Ataxia-telangiectasia (A-T), inhibiting  in stem cells and abrogates their regenerative potentials. the cGAS-STING pathway delays the aging processes by sup- Based on many research models, many intervention strate- pressing the senescence-associated secretory phenotype and the [142–145]  gies have been exploited to delay aging. ASC-derived gas- subsequent astrocyte senescence and neurodegeneration. This trointestinal organoids have also been employed in assessing result suggests the cGAS-STING pathway as a therapeutic target   + aging interventions like fasting, calorie, restriction, NAD for A-T. In a Huntington’s disease organoid model, neurodevelop- [138,148] supplementation, and inhibition of mammalian target of mental defects caused by the abnormal length of CAG repeats in [84,87] rapamycin. Using the crypts organoids as models, 24-hour the huntingtin gene (HTT) can be rescued by inhibiting a down-  fasting has been shown to enhance intestinal stem cell functions stream effector of the mutant HTT. IPSCs-derived cerebellar by activating fatty acid oxidation programs. Exogenously sup- organoid of spinocerebellar ataxia type 3 (SCA3), the second plying palmitic acid or inducing the fatty acid oxidation pro- most common polyglutamine disease after Huntington’s disease, gram with PPARδ agonists largely restores the aged intestinal has been observed spontaneous ataxin-3 aggregates in neurons,  stem cell functions in the organoids. and the SCA3 disease-associated phenotypic abnormalities in cer-  Therefore, ASC-derived organoids from different age groups ebellar neurons can be reversed by the genetic correction. With are good research models for aging. However, it is difficult to the advances in organoid technologies, we are optimistic that the obtain a large number of high-quality ASCs of different ages. use of brain organoids can provide us with valuable knowledge Although ASCs can be isolated from normal or malignant of molecular mechanisms underlying NDDs and illuminate novel human tissues by surgical resection or biopsy, the quality and ideas for developing targeted therapy. quantity of tissues can hardly be guaranteed. In addition, since some ASCs (eg, intestinal stem cells) are low in content, small Adult stem cell-derived organoids in size, possess a high nuclear-to-cytoplasmic ratio, and lack reliable surface markers, isolating ASCs directly from tissues is Gastrointestinal organoids [136,149] also one of the current technical bottlenecks. However, Even with the emergence of iPSC technologies, most gastrointes- with the development of various advanced technologies such as tinal organoids like intestinal, gastric, liver, and pancreatic ones microdissection based on high-resolution laser capture and flu- are still derived from ASC. This is likely because these tissues orescence-activated cell sorting, the technology to identify and [128,132] harbor large populations of proliferating stem-like cells.  isolate ASCs will improve, and be better applied in organoid With the declination of the number and functionality of stem research. cells during aging, tissues and organs lose the ability to maintain  homeostasis or recover from injuries (Fig. 5). Enhancing the regenerative capacity of aging stem cells will promote healthy Other organoids  aging. Therefore understanding the mechanism underly- Skin organoids ing stem cell aging is the foundation for developing effective Different from organoids derived from PSCs or ASCs discussed approaches for anti-aging therapies. above, skin organoids are generated by co-culturing skin fibro- The stem cell niche is a specialized microenvironment where blasts and keratinocytes on unique scaffolds in vitro. Skin aging stem cells reside in animals that mediate stem cell self-renewal 8 Journal of Bio-X Research Review AR ticle Figure 5. Study on ASC-derived organoid assisted stem cell exhaustion with aging. The ability of repairing tissue damages and regeneration decreases with aging because of declination of stem cells activities. The advantages of ASC-derived organoids that can reconstruct the homeostatic or regenerative conditions of their tissue of origin have been greatly reflected. Organoids generated from old ASCs show the molecular characteristics of stem cell aging in vivo, such as accumulation of SA-β-gal and age-associated epigenetic signatures. Besides, some means of aging intervention, including calorie, restriction, NAD supple- mentation and others, have also been evaluated in ASC-derived organoids. ASC=adult stem cell, mTOR=mammalian target of rapamycin, NAD =nicotinamide adenine dinucleotide, npGH=non-pituitary growth hormone, SA-β-Gal=senescence-associated β-galactosidase. Unpublished data. is caused by a combination of intrinsic and extrinsic factors, by culturing mouse podocytes and glomerular endothelial cells which eventually damage the structural integrity and physio- on the opposite sides of an extracellular matrix–coated mem-  logical function of the skin. Skin organoids generated using brane on the microfluidic chip. It has been used to reproduce fibroblasts exposed to mitomycin C can acquire characteris- the clinical manifestations of hypertensive nephropathy by  tics similar to those of the old skin, such as decreased filaggrin applying high-perfusion flows. Moreover, tissue responses expression, impaired epidermal differentiation, and reduced to inflammation, which is also known as a stress factor caus-  elastin and collagen, have been established. Skin aging model ing aging, have been successfully simulated. For instance, a 3D can also be generated by extending the skin culture time to 120 synovium-on-a-chip system consisting of patient-derived pri-  days. Prolonging the time of organoid culture in vitro as mary synovial organoids and embedded optical sensor arrays much as possible or accelerating the aging of organoids through has been developed to investigate the impact of inflammation external factors seem to be two more feasible methods to induce on the development of rheumatoid arthritis. The synovial aging. Although limitations such as the heterogeneity among cell organoids used on the chip have very similar structures to the lines used in skin organoids still exist, skin organoids provide a synovial tissue in vivo, containing a compact outermost layer novel 3D model to investigate the molecular and cellular mech- and a highly organized sublining layer with an interconnected anisms of skin aging. synoviocyte network. Exposing the 3D synovial organoids to the pro-inflammatory cytokines tumor necrosis factor-α signifi- cantly changes the tissue structure, which can be identified with Organ-on-chip  the light scatter technology. Unlike organoids that self-organize to develop into complex Aging is a systemic degeneration process that simultaneously structures, organ-on-chip is a platform that utilizes bioen- affects multiple tissues and organs. The onset and progression gineering tools to assemble matured tissue constructs into of some aging-related diseases are also mediated by inter-organ  functional units of an organ in microfabricated cell culture crosstalk. In combination with organoids, organ-on-a-chip   devices. For example, the glomerular filtration barrier, the will be a powerful tool for studying multi-organ pathologies. functional structure of the glomerulus, has been reconstructed Recapitulating these interactions via multiple mini-organs on a 9 Review AR ticle Journal of Bio-X Research chip provides an unprecedented model to study the organ cross- in organoids can be accelerated by genetic mutation or stimula- talk in vitro and shed light on the systemic mechanisms of aging. tion with exogenous factors. For example, the study of progeria syndromes, especially Hutchinson-Gilford progeria syndrome, has boosted the study of human aging due to the similarities Limitations [161–163] between progeria syndromes and biological aging. In This review highlighted studies in brain and gastrointestinal addition, external stressors, such as oxidative stress (such organoids to review the progress that has been made recently as H O and hyperoxia), genotoxic stress (such as ultraviolet 2 2 [164,165] in the application of organoids for aging research and discuss light and γ-irradiation), oncogene activation, can be used current limitations in technologies and new technologies to to manipulate organoid cultures to investigate environmental look ahead. Comparatively, the description of other organoid effects on aging. Organoids thus provide a versatile platform to models is not well-extended. Relevant publications and possible study the intrinsic and extrinsic drivers of aging and its underly- deviations are not comprehensively retrieved due to the authors’ ing cellular and molecular mechanisms. inadequate experience and expertise. Moreover, advancements in analytical methods, such as sin- gle-cell sequencing and genome engineering, will enhance our [166–168] understanding of aging. Recently, genetic screens have Conclusion and challenges been proven to be powerful tools for discovering many new The emergence of organoid models well recapitulates key key genes, such as a novel microcephaly-causing gene in brain  pathological features of aging-related diseases that cannot organoids. Using a similar strategy, more novel aging-asso- be well mimicked in 2D cell culture systems. Compared with ciated genes can be discovered by combining genetic screen-  2D cell culture systems, organoids mimic the 3D structures of ing with aging organoids. In general, the application of real organs. Organoids stand out as they better recapitulate organoids in aging research is promising. It will deepen our the complexity of organs while maintaining the accessibility understanding of aging, uncover potential molecular mark- of the traditional cell culture models. Gene mutations or fac- ers for the clinical evaluation of aging intervention and fur- tor stimulations have accelerated the onset of aging-related ther suggest strategies for combatting aging and aging-related phenotypes, making organoids a powerful tool for studying diseases. aging. Additionally, organoid culture reproduces tissue degen- eration and the eventual aging process in vitro, allowing us to Acknowledgments study the cellular and molecular features of aging at different stages and discover early molecular pathways associated with We apologize for not citing all important relevant studies in this aging. Combined with advanced technologies such as single-cell review to comply with manuscript length limitations. All the fig- sequencing, spatial transcriptomic analysis and cell-type–spe- ures were created with BioRender.com. cific genetic manipulation, organoid models will further help us analyze senescence-related spatiotemporal changes of different [155,156] Author contributions cell types and discover aging-sensitive cells. Although organoids have the potential to recapitulate key All authors participated in the literature search, manuscript pathological features of aging-related diseases, recent studies have writing, and review, and approved the final version of the revealed limitations of current organoid systems in simulating in manuscript. vivo conditions. First, compared with human organs, organoids are relatively immature. Single-cell transcriptomic analyses demon- Financial support strate that cell subtype specification is lacking from the human  forebrain organoids. Pushing the in vitro cortical organoids to This work was supported by the National Key Research and a physiological state that resembles postnatal tissue is extremely Development Program of China (Nos. 2020YFA0804000,  time-consuming. From the technical perspective, growing 2022YFA1103700, 2020YFA0112200, 2021YFF1201005), organoids to a size and shape comparable to human organs and the National Natural Science Foundation of China (Nos. extending the culture time of organoids for aging studies are still 81921006, 82125011, 92149301, 92168201, 91949209, challenging. Thus, the immaturity of organoids, compared with 92049304, 92049116, 32121001, 82192863), the Strategic adult tissues, hampers the use of organoids in modeling aging. Priority Research Program of the Chinese Academy of Sciences Methods to improve organoid maturation and structure have been (No. XDA16000000), CAS Project for Young Scientists in developed, such as bioprinting techniques, bioengineering, extra- Basic Research (Nos. YSBR-076, YSBR-012), the Program of cellular matrix modulation, vascularization, and organ-on-a-chip, the Beijing Natural Science Foundation (No. Z190019), Youth enabling extended culture times and higher resemblance to real Innovation Promotion Association of CAS (No. E1CAZW0401), [25,61,159] organs in the future. Second, the organoid culture does not the Informatization Plan of Chinese Academy of Sciences fully recapitulate the in vivo environment that organs are exposed (Nos. CAS-WX2021SF-0301, CAS-WX2022SDC-XK14, to. Thorough investigations are needed to unveil the impact of CAS-WX2021SF-0101), and the Tencent Foundation (No. extracellular matrix and growth factor selection on organoid 2021-1045). formations and functions. The absence of vascular networks and immune systems in the current organoid culture restricts the use Data availability statement of organoids in studying multifactorial diseases, such as microglia  in the brain. Third, organoids are highly variable from batch to Not applicable. batch, which increases the importance of using in vivo systems to validate findings from using organoids in vitro. Conflicts of interest Another question is how far or what aspects of aging can be mimicked by organoids. The emergence of senescent phenotypes There are no conflicts of interest. 10 Journal of Bio-X Research Review AR ticle  Fan P, Wang Y, Lu K, et al. 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Journal of Bio-X Research – Wolters Kluwer Health
Published: Mar 22, 2023
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