Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts

“…including vesicle-mediated transport (Wilmot et al., 2008), nervous system development (Lu et al., 2004) NF-kappaB transcription factor activity (Tilstra et al., 2011), regula- tion of apoptosis and inflammatory response (de Magalhães et al., 2009), and for genes previously identified to be…”

“…of Medicine, Washington University School of Medicine, St. Louis, United States; 6Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States; 7Department of Biostatistics, Fielding School of Public Health, University of”

“Epigenetic age was estimated using the published software tools (Horvath, 2013).”

“…and microRNAs in reprogrammed neurons Given the broad variability in gene expression with age in multiple cell types (Berchtold et al., 2008; Lu et al., 2004; Fraser et al., 2005; Glass et al., 2013) and a recent demonstration of main- tenance of age-associated transcriptomic changes in…”

“…of Medicine, Washington University School of Medicine, St. Louis, United States; 6Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States; 7Department of Biostatistics, Fielding School of Public Health, University of”

“DOI: 10.7554/eLife.18648 1 of 14 SHORT REPORT modeling early developmental phenotypes (Lafaille et al., 2012; Lee et al., 2009), iPSC-derived cells have been reported to be unsuitable in recapitulating phenotypes selectively observed in aged cells (Mattson and Magnus, 2006; Vera and Studer, 2015).”

“reported to increase with age, in part due to the accumulation of reactive oxygen species (ROS) (Keating, 2008; Prigione et al., 2010; Suhr et al., 2010; Cui et al., 2012).”

“…next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Huh, Christine J.; Zhang, Bo; Victor, Matheus B.; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; and Yoo, Andrew S., ,"Maintenance of age in human neurons…”

“…we previously described a reprogramming paradigm using neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), that exert reprogramming activities to directly convert human fibroblasts to specific mature neuronal subtypes (Richner et al., 2015; Victor et al., 2014; Yoo et al., 2011).”

“DOI: 10.7554/eLife.18648 1 of 14 SHORT REPORT modeling early developmental phenotypes (Lafaille et al., 2012; Lee et al., 2009), iPSC-derived cells have been reported to be unsuitable in recapitulating phenotypes selectively observed in aged cells (Mattson and Magnus, 2006; Vera and Studer, 2015).”

“DOI: 10.7554/eLife.18648 1 of 14 SHORT REPORT modeling early developmental phenotypes (Lafaille et al., 2012; Lee et al., 2009), iPSC-derived cells have been reported to be unsuitable in recapitulating phenotypes selectively observed in aged cells (Mattson and Magnus, 2006; Vera and Studer, 2015).”

“Washington University School of Medicine Digital Commons@Becker Open Access Publications 2016 Maintenance of age in human neurons generated by microRNAbased neuronal conversion of fibroblasts Christine J. Huh Washington University School of Medicine in St. Louis Bo Zhang Washington University School of Medicine in St.…”

“Cell sorting was performed on a FACSCalibur and LsrFortessa (BD Biosciences), while quantification of the percent of the population of MitoSOX positive cells was performed using FlowJo X 10.0.7r2.”

“1Department of Developmental Biology, Washington University School of Medicine, St. Louis, United States; 2Program in Molecular and Cellular Biology, Washington University School of Medicine, St. Louis, United States; 3Program in Neuroscience, Washington University School of Medicine, St. Louis, United States; 4Depart…”

“The utility of the epigenetic clock method has been demonstrated in applications surrounding cognitive function (Levine et al., 2015), obesity (Horvath et al., 2014), Down syndrome (Horvath et al., 2015b), HIV infection (Horvath and Levine, 2015), and Parkinson’s disease (Horvath and Ritz, 2015).”

“generated by microRNA-based neuronal conversion of fibroblasts”

“‘Aging’ transcriptome and microRNAs in reprogrammed neurons Given the broad variability in gene expression with age in multiple cell types (Berchtold et al., 2008; Lu et al., 2004; Fraser et al., 2005; Glass et al., 2013) and a recent demonstration of main- tenance of age-associated transcriptomic…”

“Oxidative stress has been reported to increase with age, in part due to the accumulation of reactive oxygen species (ROS) (Keating, 2008; Prigione et al., 2010; Suhr et al., 2010; Cui et al., 2012).”

“…at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Huh, Christine J.; Zhang, Bo; Victor, Matheus B.; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; and Yoo, Andrew S., ,"Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts."”

“The utility of the epigenetic clock method has been demonstrated in applications surrounding cognitive function (Levine et al., 2015), obesity (Horvath et al., 2014), Down syndrome (Horvath et al., 2015b), HIV infection (Horvath and Levine, 2015), and Parkinson’s disease (Horvath and Ritz, 2015).”

“…which is virtually unchanged (Figure 4C), in contrast to the progressive increase in length commonly observed with iPSC reprogramming, where telomeres reach a plateau of around 12–14 kilobases after a few cellular passages (Agarwal et al., 2010; Batista et al., 2011; Marion et al., 2009).”

“The utility of the epigenetic clock method has been demonstrated in applications surrounding cognitive function (Levine et al., 2015), obesity (Horvath et al., 2014), Down syndrome (Horvath et al., 2015b), HIV infection (Horvath and Levine, 2015), and Parkinson’s disease (Horvath and Ritz, 2015).”

“The utility of the epigenetic clock method has been demonstrated in applications surrounding cognitive function (Levine et al., 2015), obesity (Horvath et al., 2014), Down syndrome (Horvath et al., 2015b), HIV infection (Horvath and Levine, 2015), and Parkinson’s disease (Horvath and Ritz, 2015).”

“cesses including inflammation, senescence, metabolism and telomerase activity (Kondo et al., 2016; Qin et al., 2012; Fang et al., 2010).”

“…of Medicine, Washington University School of Medicine, St. Louis, United States; 6Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States; 7Department of Biostatistics, Fielding School of Public Health, University of”

“…which is virtually unchanged (Figure 4C), in contrast to the progressive increase in length commonly observed with iPSC reprogramming, where telomeres reach a plateau of around 12–14 kilobases after a few cellular passages (Agarwal et al., 2010; Batista et al., 2011; Marion et al., 2009).”

“generated by microRNA-based neuronal conversion of fibroblasts”

“…in age from 3 days to 96-year old were transduced with a lentiviral preparation of the Doxycline-inducible synthetic cluster of miR-9/9* and miR-124 (miR-9/9*-124), alongside transcription factors CTIP2, DLX1, DLX2, and MYT1L as previously described (Richner et al., 2015; Victor et al., 2014).”

“Oxidative stress has been reported to increase with age, in part due to the accumulation of reactive oxygen species (ROS) (Keating, 2008; Prigione et al., 2010; Suhr et al., 2010; Cui et al., 2012).”

“Moreover, reprog- rammed neurons were analyzed by Comet Assay, a single-cell gel electrophoresis technique that assesses DNA damage accumulation (Singh et al., 1990).”

“…Washington University School of Medicine, St. Louis, United States; 2Program in Molecular and Cellular Biology, Washington University School of Medicine, St. Louis, United States; 3Program in Neuroscience, Washington University School of Medicine, St. Louis, United States; 4Department of…”

“…at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Huh, Christine J.; Zhang, Bo; Victor, Matheus B.; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; and Yoo, Andrew S., ,"Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts."”

“…of Medicine, Washington University School of Medicine, St. Louis, United States; 6Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States; 7Department of Biostatistics, Fielding School of Public Health, University of”

“While the exact role of these microRNAs in aging is unknown, miR-10a and miR-497 have been previously implicated in aging-associated cellular pro- cesses including inflammation, senescence, metabolism and telomerase activity (Kondo et al., 2016; Qin et al., 2012; Fang et al., 2010).”

“…in age from 3 days to 96-year old were transduced with a lentiviral preparation of the Doxycline-inducible synthetic cluster of miR-9/9* and miR-124 (miR-9/9*-124), alongside transcription factors CTIP2, DLX1, DLX2, and MYT1L as previously described (Richner et al., 2015; Victor et al., 2014).”

“The data were normalized using the ’Noob’ normalization method (Triche et al., 2013).”

“Recently, experimental manipulations to accelerate aging in iPSC-derived cells have been explored, for instance, by overexpressing progerin, a mutant form of lamin A observed in progeria syndrome, to force the detection of agerelated pathophysiology of neurodegenerative disease (Arbab et al., 2014; Cornacchia and Stud…”

“…population doubling level (PDL) to elimi- nate any confounding variability introduced by sequential passaging (Campisi and d’Adda di Fagagna, 2007; Pazolli and Stewart, 2008), then subsequently transduced with miR-9/9*-124 with CTIP2, DLX1/2, and MYT1L (CDM) following our established protocol…”

“…we previously described a reprogramming paradigm using neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), that exert reprogramming activities to directly convert human fibroblasts to specific mature neuronal subtypes (Richner et al., 2015; Victor et al., 2014; Yoo et al., 2011).”

“While the exact role of these microRNAs in aging is unknown, miR-10a and miR-497 have been previously implicated in aging-associated cellular pro- cesses including inflammation, senescence, metabolism and telomerase activity (Kondo et al., 2016; Qin et al., 2012; Fang et al., 2010).”

“…we previously described a reprogramming paradigm using neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), that exert reprogramming activities to directly convert human fibroblasts to specific mature neuronal subtypes (Richner et al., 2015; Victor et al., 2014; Yoo et al., 2011).”

“Recently, experimental manipulations to accelerate aging in iPSC-derived cells have been explored, for instance, by overexpressing progerin, a mutant form of lamin A observed in progeria syndrome, to force the detection of agerelated pathophysiology of neurodegenerative disease (Arbab et al., 2014; Cornacchia and Stud…”

“➌ Additionally, DNA lesions can accumulate via errors in transcription or in the removal of epigenetic methylation markers, and persistent lesions fundamentally alter metabolism due to bioenergetic stress; ➍ this leads to changes at the mitochondrial level, which in turn increase the amount of reactive oxygen species…”

“, 2018) and observed increased DNA damage repair in old and AD iNs (Huh et al., 2016; Mertens et al., 2021), AD iNs showed a higher demand for NAD that was”

“Our results on spinal cord FibiMNs are in general agreement with previous studies showing that aging features are retained in directly reprogrammed brain neurons (Mertens et al., 2015; Huh et al., 2016).”

“These direct conversion modalities may prove to be invaluable in the study of late-onset neurodegenerative diseases because the original age of human fibroblasts is maintained in converted neurons (Huh et al., 2016; Mertens et al., 2015) in contrast to the cellular rejuvenation observed in induced pluripotent stem cel…”

“Direct neuronal reprogramming of human cells maintains many hallmarks of aging, such as epigenetic marks, nuclear lamina, and mitochondrial dysfunction (Horvath, 2013; Huh et al., 2016; Kim et al., 2018; Mertens et al., 2015).”

“In contrast, fibroblasts directly reprogrammed into neurons retain their aging characteristics such as DNA methylation (Huh et al., 2016; Maherali et al., 2007; Mertens et al., 2015).”

“study of age-related neurodegenerative diseases (Huh et al., 2016; Mertens et al., 2015; Tang et al., 2017).”

“In addition, recent studies have also demonstrated that the resulting iNs, unlike iPSCs, maintain the aging signature of the donor, making iNs ideal candidates for modeling neuronal pathology in late-onset diseases (Mertens et al , 2015; Huh et al , 2016).”

“Importantly, avoiding the iPSCs stage, transdifferentiated iNs were shown to conserve the agerelated epigenetic landscape and other cellular properties from the cell of origin [138, 140].”

“Recently, an HD patient fibroblast model from which medium spiny neurons (MSNs) are generated through direct neuronal conversion (Huh et al., 2016; Mertens et al., 2015) was shown to demonstrate an aggregation phenotype of endogenous mHtt, in the absence of extreme polyQ expansions that are rarely observed in the pati…”

“These results underscore the potential of 3D neuronal reprogramming to model age-dependent A β deposition in LOAD neurons.”

“Upregulated in the hippocampus of old mice [167]; downregulated in old induced striatal medium spiny neurons [168].”

“Additional commonalities of senescence and aging include alteration of cellular morphology, metabolic changes, loss of differentiation potential, activation of the p53/p21CIP1 and p16INK4A/pRb signaling pathways, increased senescenceassociated β-galactosidase activity (SA-β-gal), formation of senescence-associated het…”

“…recent studies have shown that neurons directly reprogrammed from fibroblasts without the use of pluripotency factors retain ageing signatures compared to induced pluripotent stem cells (iPSC)-derived neurons (Huh et al., 2016; Mertens et al., 2015; Tang et al., 2017; Victor et al., 2018).”

“…cells DMEM/F12 BDNF, GDNF, heparin, IGF1, RA, SHH, N2 cAMP, purmorphamine Li et al., 2008 Human adipose tissue derived stem cells DMEM FGF2 Forskolin Jang et al., 2010 #71 Human mesenchymal stem cells NSA- basal medium Br-cAMP, dorsomorphin, RG108, rolipram TSA Funk and Alexanian, 2013 Human…”

“Moreover, they show that the aging signature is maintained through their microRNA expression profile and increased oxidative stress levels (Huh et al., 2016).”

“Such methods of direct conversion have been shown to maintain multiple epigenetic age-associated signatures when converted from fibroblast to neuron [Huh et al., 2016].”

“The generation of human hippocampal neurons through direct reprogramming of fibroblasts, or directed differentiation of induced pluripotent stem cells, holds promise to delineate the neurobiological changes in aging at a cellular level [224, 225].”

“These iNs retain many of the epigenetic marks of the source tissue [44, 45] which can capture the epigenetic signature of aging.”

“Stem Cell Repor This is an open access article under the C chondrial defects (Huh et al., 2016; Kim et al., 2018; Mertens et al., 2015, 2021; Tang et al., 2017).”

“Importantly, neurons produced from fibroblasts maintain age-related epigenetic marks, which makes them particularly suited for modeling later-onset neurodegenerative disorders.(104,105) In general, TF-based reprogramming of stem cells involves two simple steps: first, infection of stem cells with a combination of TFs…”

“, 2013), thereby retaining the age signatures of starting fibroblasts including the epigenetic clock (Horvath, 2013), ageassociated changes in transcriptome and microRNAs, reactive oxygen species (ROS) levels, DNA damage and telomere lengths (Mertens et al., 2015; Huh et al., 2016; Tang et al., 2017).”

“The “bioreactors” used in culture may also improve the health of the cells (Marion et al., 2009; Corti et al., 2012; Baxter et al., 2015; Huh et al., 2016; Mertens et al., 2018).”

“, 2008), directly converted cells maintainmany features of aging (Huh et al., 2016;Mertens et al., 2015).”

“Indeed, several studies have shown that iNs appear to retain transcriptomic and epigenetic characteristics similar to the aged donor somatic cells (Huh et al., 2016; Mertens et al., 2015).”

“Fibroblast-derived iNs preserve features of human aging including transcriptomic, epigenetic, mitochondrial and nuclear pore changes (Herdy et al. 2019; Huh et al. 2016; Kim et al. 2018; Mertens et al. 2015).”

“also showed that direct conversion using this protocol preserves age-related epigenetic and cellular signatures from the donor, similar to established transcription factor-only methods (Huh et al., 2016).”

“In addition, iNs, unlike iPSCs, maintain the ageing [30] and the epigenetic marks of the donor [31,32], making them excellent candidates for modelling neuronal pathophysiology in age-related disorders.”

“Finally, because reprogramming of somatic cells to hiPSCs erases epigenetic marks (12) we tested whether leptomeningeal cells are a viable starting cell source for direct neuron conversion.”

“Besides transcriptomic signatures, iN preserve the donor cell's age‐associated epigenetic memory and other aging hallmarks such as shortened telomere lengths, elevated ROS levels and accumulated DNA damage (Huh et al., 2016).”

“…is an essential part of the cellular reprogramming process; while important epigenetic markers associated with complex genetic diseases may be lost during hiPSC reprogramming, this does not seem to occur to the same extent during neuronal induction (Huh et al., 2016; Mertens et al., 2016).”

“Recent developments in direct neuronal transdifferentiation from adult somatic cell types appear to preserve aspects of cellular age (Abernathy et al. 2017; Huh et al. 2016; Victor et al. 2018; Yoo et al. 2011) and may facilitate the development of more accurate in vitro models of TBI pathogenesis.”

“Using a miRNAbased cellular reprogramming approach, human fibroblasts obtained from donors ranging from neonates to near centenarians could be efficiently converted into neurons that retain multiple age-associated signatures (Huh et al., 2016).”

“…whether it would be feasible to reduce the RCAN1 adult fibroblasts to neurons has been shown to propagate chronological age-related characteristics such as epigenetic cellular age stored in starting fibroblasts, thereby generating neurons that mimic the epigenetic age of fibroblast donors 6 .”

“…the differentiation process [103]; and 6) reprogramming into iPSCs can reset cellular age of lineage-speci fi c derived cells, such as neurons [104], and/or induces mitochondrial rejuvenation [105], thereby no longer exhibiting the original pathophysiological characteristics of a PMD.…”

“It is hypothesized that the conservation of epigenetic markers may be achieved through direct reprogramming strategies [43] if donor-specific epigenetic profiles, such as with aging [44, 45], are of interest.”

“In addition, iNs, unlike iPSCs, maintain the ageing (Mertens et al., 2015) and the epigenetic marks of the donor (Horvath, 2013; Huh et al., 2016), making them excellent candidates for modelling neuronal pathophysiology in age-related disorders.”

“A recent study found that the epigenetic age (or more precisely termed ‘DNA methylation (DNAm) age’), a distinctive pattern of DNA methylation marks from which a donor’s age can be predicted, was the same between iNs and their parental fibroblast [30].”

“While reprogramming of fibroblasts into hiPSCs erases age-related epigenetic marks and gene expression profiles, these can be at least partially maintained by inducing patient somatic cells directly into neurons (Mertens et al., 2015; Huh et al., 2016).”

“The lack of cellular rejuvenation, shorter reprogramming durations and reduced labor required for iN generation are attractive for disease modeling.(14,15) However, iNs do not follow successive stages of neurodevelopment.”

“Unlike iPSC-derived neurons, iNs show mature neuronal 5-hmC methylation and show transcriptomic aging signatures and preservation of their donors’ DNAm clock ages (Table 1)(Herdy et al., 2019; Huh et al., 2016).”

“…(MET) (Li et al. 2010; Samavarchi-Tehrani et al. 2010), (4) a metabolic remodeling from oxidative phosphorylation to glyco-lysis-based metabolism (Zhang et al. 2016; Zhu et al. 2024), (5) the activation of early pluripotency-associ-ated genes (such as alkaline phosphatase and SSEA-1 in mouse;…”

“Besides being an invaluable tool to create patient and disease-specific neurons for modeling of age-associated neurological disorders ( 11 – 15 ), direct reprogramming offers a promising nonpluripotent cell source for personalized transplantation-based therapies for neurodegenerative diseases.”

“Excitingly, a recent technological advancement that can help overcome this limitation is the direct differentia - tion of fibroblasts to desired neuronal populations (143, 144).”

“2; Horvath 2013; Huh et al. 2016; Sheng et al. 2018).”

“Specifically, common findings in humans, animals, and reprogrammed neurons have demonstrated an age-related decrease in the antioxidative defense system [42,54,55] together with heightened ROS levels [56,57], leading to an augmentation in oxidative stress [58,59].”

“In contrast, direct conversion of fibroblasts into induced neurons (iNs) circumvents the pluripotent intermediate state, and it has been demonstrated that iNs retain the transcriptomic and epigenetic age of the starting cells [29, 35].”

“Moreover, in iMSNs, the length of telomeres is unaltered, and it corresponds to the telomere length of the starting fibroblasts, which is related to the donor’s age [4].”

“…Junien, 1993; Sabouri et al., 1993; Shaw et al., 1993; Jansen et al., 1996; Benders et al., 1997; Berul et al., 1999; Eriksson et al., 2000, 2001; Inukai et al., 2000; Klesert et al., 2000; Mounsey et al., 2000; Sarkar et al., 2000; Depardon et al., 2001; Coffee et al., 2002) YES (Tabolacci et…”

“Direct neuronal reprogramming, by which a neuron is formed via direct conversion from a somatic cell without going through a pluripotent intermediate stage, allows the possibility to generate patient-derived neurons that maintain the aging and epigenetic signatures of the donor [128, 129].”