Method for inducing artificial aging in stem cells and use of said cells

Abstract

The present invention relates to a method of transiently inducing aging in stem cells or somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising introducing one or more aging-inducing factors into the cell or exposing the cell to one or more exogenous aging-inducing factors for a period of time, the one or more aging-inducing factors are then removed or reduced, such as before or after forward programming of the iPSC, resulting in a reversal of the one or more aging phenotypes in the cell. Also provided are artificially aged stem cells or somatic cells derived from iPSC by forward programming, and their use in methods of identifying genes or combinations of genes involved in aged phenotype reversal or non-aged phenotype maintenance in cells. Also provided are methods of identifying a gene or combination of genes involved in aging phenotype reversal or non-aging phenotype maintenance in a cell comprising introducing one or more aging-inducing factors into the cell or exposing the cell to one or more exogenous aging-inducing factors for a period of time, and measuring the aging phenotype of the cell. In particular, methods of identifying a gene or combination of genes involved in aging phenotype reversal or non-aging phenotype maintenance in a cell optionally include loss of function, inhibition, knockout, function acquisition, or combination screening. Also provided is a method of regenerating somatic cells comprising altering the expression and / or activity of one or more genes identified by the methods described herein.

Description

Technical Field

[0001] The present invention relates to a method for transiently inducing aging in stem cells or somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising introducing one or more aging inducing factors into cells or exposing cells to one or more exogenous aging inducing factors for a period of time, such as before or after the forward programming of iPSCs, and then removing or reducing the one or more aging inducing factors, thereby causing the reversal of one or more aging phenotypes in cells. Also provided are artificially aged stem cells or somatic cells derived from iPSCs by forward programming, and their use in methods for identifying genes or gene combinations involved in reversing aging phenotypes or maintaining unaged phenotypes in cells. Also provided are methods for identifying genes or gene combinations involved in reversing aging phenotypes or maintaining unaged phenotypes in cells, the method comprising introducing one or more aging inducing factors into cells or exposing cells to one or more exogenous aging inducing factors for a period of time, and measuring the aging phenotype of cells. In particular, the method of identifying a gene or gene combination involved in the reversal of an aging phenotype or the maintenance of an unaged phenotype in a cell optionally includes performing loss-of-function, inhibition, knockout, gain-of-function or combined screening. Also provided is a method of regenerating somatic cells, comprising changing the expression and / or activity of one or more genes identified by the methods described herein. Background Art

[0002] Aging is a common risk factor for a variety of diseases from cancer to dementia (Niccoli and Partridge (2012) Curr. Biol.). However, many findings suggest that the aging trajectory can be modulated, for example through dietary interventions (Longo and Mattson (2014) Cell Metab.; Longo et al. (2021) Nat. Ageing), drugs (Kennedy and Pennypacker (2014) Tranl. Res.; Guilbert et al. (2021) Methods), and genetic manipulations (Simpson et al. (2021) Clin Epigenetics; Zhang et al. (2020) Nat Rev Mol Cell Biol). Most importantly, reprogramming somatic cells into induced pluripotent stem cells can reverse many features of aging (Lapasset et al. (2011) Genes Dev.). Therefore, there are mechanisms that can modulate aging, which deserves further investigation.

[0003] Although the aging process is still poorly understood, it affects cells in many ways. Otin et al. (2013) Cell has provided a comprehensive review of "The Hallmarks of Aging", which include mitochondrial changes, senescence, altered intracellular communication, genomic instability, telomere shortening, epigenetic changes, and dysregulation of nutritional pathways. These may lead to changes in cellular function, such as a reduced ability of immune cells to investigate carcinogenic events, which ultimately leads to a large number of age-related diseases.

[0004] Stem cells provide an expandable model system for studying species biology. However, due to the embryonic nature of pluripotent stem cells, particularly cell regeneration during iPSC reprogramming, using pluripotent stem cells (PSC) such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) technology to model late-onset disorders is challenging (Lapasset et al. (2011) Genes Dev.; Maherali et al. (2007) Cell Stem Cell; Studer et al. (2015) Stem Cell, 16:591-600).

[0005] In addition, the characteristics of aging are generally related to the degenerative process associated with the disease. Independently, they are related to the gradual degradation of structure and function. Some of these marks have been identified as nuclear blebbing and folding, shortening of dendrite length (in terms of neurons), telomere wear, reduced protein homeostasis and accumulation of DNA damage and mitochondrial reactive oxygen species (ROS) (Lopez-Otin et al. (2013) Cell; Miller et al. (2013) Cell Stem Cell; Studer et al. (2015) Stem Cell). In addition, changes in gene expression profiles have been observed in neurons (Arancio (2019) Gero Science; Mertens et al. (2015) Cell Stem Cell, 17: 705-718) and human brain (Dillman et al. (2017) Sci. Rep.).

[0006] These findings further confirm the necessity of generating reliable iPSC models of human diseases, and thus inspire two major strategies for inducing cell aging. The first approach involves manipulating the cellular environment by introducing toxic stressors such as reactive oxygen species (Davalli et al. (2016) Oxid. Med. Cell. Longev.) or by inflammatory cytokines to simulate the general overall inflammatory state associated with aging. The second approach involves introducing intrinsic changes into cells to induce aging. This is achieved by pharmacologically inhibiting telomerase to promote telomere shortening, which is an enzyme involved in maintaining telomere length (Vera et al. (2016) Cell Rep.). In this study, similar age-related and disease-related phenotypic changes were reported in brain dopaminergic neurons in Parkinson's disease, such as increased mitochondrial ROS production, DNA damage, and loss of tyrosine hydroxylase expression. However, the study is still in its preliminary stages because no effects on other cellular processes were observed.

[0007] Therefore, there is a need to develop systematic approaches to identify genes and regulatory networks that contribute to aging and the reversal of aging in iPSCs, which can subsequently be applied to somatic cells. Summary of the invention

[0008] According to a first aspect of the present invention, there is provided a method for transiently inducing aging in stem cells, the method comprising:

[0009] (i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0010] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0011] According to another aspect of the present invention, there is provided a method for transiently inducing aging in somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising:

[0012] (i) before or after forward programming of iPSCs, introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0013] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0014] In certain embodiments, removing or reducing one or more aging-inducing factors in step (ii) comprises actively removing or reducing, thereby resulting in reversal of one or more aging phenotypes in the cell.

[0015] In another aspect, artificial aged stem cells produced by the methods described herein or somatic cells derived from iPSCs by forward programming are provided.

[0016] In a further aspect, there is provided use of artificial aged stem cells as defined herein and / or somatic cells derived from iPSCs by forward programming in a method for identifying a gene or a combination of genes involved in reversal of an aged phenotype or maintenance of a non-aged phenotype in a cell.

[0017] According to another aspect of the present invention, there is provided a method for identifying a gene or gene combination involved in reversing an aging phenotype or maintaining a non-aging phenotype in a cell, the method comprising the following steps:

[0018] i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time;

[0019] ii) optionally performing loss-of-function, inhibition, knockout, gain-of-function or a combination screening in said cell;

[0020] iii) measuring the senescence phenotype of said cells; and

[0021] iv) identifying genes or gene combinations as involved in reversal of an aged phenotype or maintenance of a non-aged phenotype when, upon loss-of-function, inhibition, knockout, gain-of-function or combination screening, the aged phenotype is altered and / or its progression is altered in cells compared to when the cells have not been screened.

[0022] In another aspect of the present invention, there is provided a method of regenerating a somatic cell comprising altering the expression and / or activity of one or more genes identified by the methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1:Aging and regeneration of aged hiPSC.A) Schematic diagram of experimental steps for inducing aged iPSC and aged cortical glutamatergic neurons, which have been "aged" before (i) or after (ii) iPSC is converted into neurons.BC) hiPSC cell lines are generated in two genetic backgrounds, overexpressing progerin (progerin) only under doxycycline control (B), and targeting cells with NGN2 and progerin induction boxes in a dual system using TetOn and Cumate systems for overexpression (C).DF) NGN2 iPSC (D), NGN2-progerin iPSC (E) and progerin iPSC (F) are cultured with or without cumate or dox (doxycycline) to overexpress progerin and artificial aging cells. Aged iPSCs retained their pluripotency markers (i.e., Nanog and OCT4) after inducing GFP-progerin for 5 days. However, lamin A and GFP staining show abnormal nuclear membrane morphology in aged iPSCs. After passage of aged iPSCs and cessation of progerin expression, cells regained their morphology (E and F, 1st and 2nd divisions). GH) Compared to non-aged iPSCs, it was found that the nuclei of aged NGN2-progerin iPSCs (G) and aged progerin iPSCs (H) were no longer as round and more elongated. Aged iPSCs, in which progerin was no longer expressed, reversed their morphological abnormalities after the 1st passage / division. Data are expressed as mean ± SEM, one-way ANOVA. Scale bar = 25 μm. NGN-, no cumate; NGN+, with cumate; N2P-, no cumate; N2P+, with cumate; 1st / 2nd division, aged N2P / progerin iPSCs 1st or 2nd division after aging. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 2:Morphological changes of progerin after overexpression in KOLF progerin iPSC. A) Progerin iPSCs were cultured with or without dox to overexpress progerin. Aged iPSCs retained their pluripotency markers Nanog and OCT4 after 5 days of induction of GFP-progerin. GFP staining shows abnormal nuclear membrane morphology of aged iPSCs. After passage of aged iPSCs, when progerin expression stops, cells recover their morphology. Scale bar = 25 μm. B) Morphological analysis of circularity, roundness and size of the nucleus after overexpression of progerin and after the first and second divisions after aging. The nuclear circularity and roundness in aged cells are reduced, but when progerin expression stops, it spontaneously returns to normal morphology after the first division after aging. Interestingly, after aging, the size of the nucleus is also significantly reduced. Data are expressed as mean ± SEM, one-way ANOVA. **p<0.01, ****p<0.0001.

[0025] Figure 3 : Aging iPSCs with reduced heterochromatin expression and increased DNA damage. AC) After aging induced by cumate in hiPSCs, the peripheral heterochromatin H3K9me3 mark was significantly downregulated. However, after stopping progerin expression for 1 generation, cells began to show recovery of H3K9me3 expression. Scale bar = 25μm. Data are expressed as mean ± SD, one-way ANOVA. DE) In aged iPSCs, H3K9me3 was significantly reduced after overexpression of progerin by dox, but returned to normal levels after the 1st and 2nd divisions after aging. Scale bar = 25μm. Data are expressed as mean ± SD, one-way ANOVA. F) Aged iPSCs show double-stranded DNA breaks (based on γH2AX staining), which are no longer present after the 1st division after aging. Scale bar = 10μm. G) The number of foci was counted based on γH2AX expression. Aged iPSCs showed the highest number of foci ≥ 5, whereas non-senescent cells and cells aged by transient progerin expression after the 1st and 2nd divisions had significantly fewer foci. Data are presented as mean ± SEM, one-way ANOVA. NGN-, no cumate; NGN+, with cumate; N2P-, no cumate; N2P+, with cumate; 1st / 2nd division, aged N2P / progerin iPSCs after 1st or 2nd division. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0026] Figure 4: Immunostaining of heterochromatin and DNA damage markers in Kolf aged iPSCs. A) According to immunocytochemistry, the expression of the peripheral marker H3K9me3 of heterochromatin appears to be reduced in transiently aged iPSCs, but returns to normal levels after the first and second divisions after aging. Scale bar = 25 μm. B) Aged iPSCs show double-stranded DNA breaks (based on γH2AX staining), which decrease after the first division after aging. Scale bar = 10 μm. C) Quantitative analysis of H3K9me3 expression. Data are expressed as mean ± SEM, one-way ANOVA. D) The number of lesions is counted based on γH2AX expression. Aged iPSCs show the highest number of lesions ≥ 5, while the number of lesions in iPSCs that are not aged and transiently aged after the first and second divisions is significantly less. Data are expressed as mean ± SEM, one-way ANOVA. **p<0.01, ****p<0.0001, ns=not significant.

[0027] Figure 5 : Senescence in aged iPSCs. A) Phase images of Bob and Kolf non-aged and aged iPSCs after β-galactosidase (β-gal) staining. Scale bar = 250 μm. B) Based on quantitative analysis, aged iPSCs showed a significant increase in β-gal staining compared to non-aged iPSCs. Data are expressed as mean ± SD, one-way ANOVA. *p < 0.05, **p < 0.01.

[0028] Figure 6 :The initial NGN2-presenilin construct is used in the OPTi-OX system to generate aged iNeurons. A) The initial NGN2-presenilin reprogramming box is used to overexpress NGN2 and presenilin under the control of the TetOn system. NGN2 and GFP-presenilin are separated by the P2A site and expressed simultaneously. B) Phase and confocal images of non-aged (iN) and aged iNeurons (iaN) 3 days after induction. GFP-presenilin expression is visible (left). Nuclear folding and blebbing are observed by lamin A staining (right). Scale bar = 25μm. C) According to RT-PCR analysis, lamins A and C are upregulated after overexpression of presenilin in aged iNeurons. D) Based on oxygen consumption rate (OCR) measurements, aged iNeurons show reduced mitochondrial function. E) Viability assays on days 1 and 3 after induction show a large number of cell deaths in aged iNeurons.

[0029] Figure 7 :Development of dual induction system (OPTi-OX) 2, to artificially age iPSCs and iNeurons. A) Workflow for inducible NGN2 / NGN2-progerin expression in hiPSCs targeting the human ROSA26 and AAVS1 loci using the Tet-ON and Cumate systems. Cas9n, D10A nickase mutant Cas9 endonuclease; ZFN, zinc finger nuclease; rtTA, reverse tetracycline transactivator; CymR, cumate repressor. B) Expression of NGN2 / NGN2-progerin in hiPSCs using the Tet-ON and Cumate systems. 2 Schematic representation of four outcomes after generating dual GSH-targeted inducible NGN2 / NGN2-presenilin hiPSCs in the (Tet-ON / Cumate) system: Clonal cell lines were sorted according to the number of successfully targeted human ROSA26 and AAVS1 loci alleles. C) Two expression cassettes were designed, with the first construct directing NGN2 and presenilin transgenes in the same manner, while the second construct directed the two transgenes in opposite directions to minimize potential leakage. D) Genotyping results of selected human ROSA26-CAG-rtTA-CymR-targeted (left) and AAVS1-NGN2-presenilin-retargeted hiPSCs (right). Heterozygous cell lines (for the ROSA26 locus) and homozygous cell lines (for the AAVS1 locus) for each allele are shown. WT: wild-type hiPSC; B.N2P: Bob NGN2-presenilin; K.N2P, KOLF2 NGN2-presenilin; NT: no template control. E) Phase images of the three cell lines tested (Bob NGN2, Bob NGN2-presenilin, KOLF2 NGN2-presenilin) ​​7 days after induction. No differences in neuronal growth were observed between these cell lines. Scale bar = 100 μm. FG) Phase images (F) and immunocytochemistry (G) of non-aged (dox only) and aged iNeurons (dox+cumate) 1 week after induction. After overexpression of presenilin at different concentrations, folding and blebbing of the nuclear membrane were observed by GFP-presenilin expression. Scale bar = 50 μm upper figure; 10 μm lower figure. H) Cumate dose response assay. Data are expressed as mean ± SD, one-way analysis of variance. I) Viability assay. Data are expressed as mean ± SD, Student t-test.

[0030] Figure 8: Pre-aged and post-aged iNs exhibit abnormal nuclear morphology. AB) Colocalization of GFP-presenilin and lamin A expression in Bob-N2P and KOLF-N2P aged iNeurons at D14 (A) and D21 (B) after induction, showing folded and ruptured nuclear morphology. No expression was observed in control iNeurons (Bob-NGN2). Scale bars: left = 25 μm; right = 10 μm. CD) After overexpression of presenilin at D14 (C) and D21 (D) after induction, the lamina associated protein LAP2α was found to be significantly downregulated. Scale bar = 25 μm. EF) Quantitative analysis of LAP2α expression at D14 (E) and D21 (F) after induction. Data are expressed as mean ± SEM. One-way ANOVA. GH) At D14 (G) and D21 (H) after induction, the circularity and roundness of the nucleus were found to be affected in aged iNeurons compared to non-aged iNeurons. Data are presented as mean ± SEM. One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0031] Fig. 9: Degeneration of neurites after overexpression of progerin. A) Based on immunocytochemistry detection of β3-tubulin and MAP2, overexpression of progerin leads to shortened complete dendrite length in aged iNeurons (iaN / aiN) compared with non-aged iNeurons (iN) at D7 (upper), D14 (middle) and D21 (lower) after induction. Scale bar = 100 μm phase, 25 μm ICC. BC) Based on quantitative analysis of MAP2 staining, aged iNeurons (iaN / aiN) have significantly shorter dendrites compared with non-aged iNeurons at D14 (B) and D21 (C) after induction. Data are expressed as mean ± SEM, one-way ANOVA. DE) Compared with the more widely distributed non-aged iNeurons, the diameter (D) of D21 aged iNeurons is significantly lower, and the distribution of neurite diameter (E) is narrower, concentrated around 300 nm. Nonlinear regression, Gaussian distribution. Mean ± SEM, one-way ANOVA. F) After induction of iNeuron aging, the mRNA levels of cytoskeletal (β3-tubulin, MAP2) and synaptic (synaptophysin, synapsin-1, PSD95, vGlut2) related neuronal markers were downregulated. Data are expressed as mean ± SD, all data are relative to PBGD1, one-way ANOVA. G) Based on immunocytochemistry, synapsin-1 was downregulated after progerin overexpression. Scale bar = 25 μm. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns = not significant.

[0032] Fig.10 : Presenilins themselves, not cumate, are responsible for the aging phenotype. A) Comparison of Bob NGN2 and Bob N2P iNs treated with or without cumate. RT-PCR analysis confirmed that cumate was not responsible for the changes seen in gene expression, namely the downregulation of MAP2 and synapsin-1. Data are expressed as mean ± SD, all data are relative to PBGD1, student t-test. B) Immunocytochemistry of Bob N2P and BobNGN2 iNeurons treated with (middle and bottom panels) or without (top panels) cumate. Overexpression of presenilins leads to a reduction in dendritic morphology and H3K9me3 expression. Scale bar = 25 μm. **p<0.01, ns = not significant.

[0033] Fig.11: Peripheral heterochromatin marker expression and DNA damage in aged iNeurons. AB) Overexpression of progerin in aged iNeurons (iaN / aiN) resulted in decreased expression of H3K9me3 (A) and HP1γ (B) at D14 (AB, top) and D21 (AB, bottom) after induction. Scale bar = 25 μm. CF) H3K9me3 and HP1γ were significantly downregulated at D14 (C, E) and D21 (D, F) after induction compared to control non-aged iNeurons. Data are expressed as mean ± SEM, one-way ANOVA. G) DNA damage observed in aged iNeurons of two cell lines (Bob and Kolf) based on γH2AX staining after progerin overexpression. Scale bar = 15 μm. HI) The number of foci >5 per cell was higher in the aged iNeuron group, with 18% and 12% of cells having ≥3 foci for the Bob and Kolf cell lines, respectively. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

[0034] Fig.12: Mitochondrial function and electrophysiology of aged iNeurons. AC) Oxygen consumption rate (OCR) of unaged (iN) and aged (iaN / aiN) iNeurons at D14 was measured. Compared with unaged iNeurons, post-aged (AB) and pre-aged (B) iNeurons showed a decrease in mitochondrial function. Data are expressed as mean ± SD. D) The basal respiratory level of the aged group was significantly lower than that of the unaged group. Data are expressed as mean ± SD. One-way ANOVA. E) Viability assay after OCR measurement confirmed that the decrease in OCR was due to overexpression of progerin rather than cell death. Live cells were normalized to control (Bob NGN2 or Bob N2P iNeurons treated with dox only). F) Schematic diagram of the multi-electrode array (MEA) (Axion) used in this study. Each well contains 16 electrodes. G) Electrophysiological activity recordings on MEA of non-aged iNeurons (iN) and aged iNeurons (iaN / aiN) with and without astrocytes. At D14 after induction, the mean firing rate of the aged group was significantly reduced, whereas at D21, its activity was similar to that of the non-aged group. Data are presented as mean ± SEM. One-way ANOVA. H) At D21 after induction, there was no difference in burst duration between the aged and non-aged groups, indicating no effect on neural network communication. Data are presented as mean ± SEM. One-way ANOVA. ****p<0.0001, ns=not significant.

[0035] Fig.13 : Transcriptome profiles of aging cells after overexpression of progerin. A) Principal component (PC) analysis shows tight clustering of control non-aging iPSC (middle) group and iNeuron (right) group. B) Overlap of differentially expressed genes (two left graphs) and aging-related genes (two right graphs). This analysis provides a list of genes that can be used for CRISPR screening to identify gene regulatory networks that lead to aging phenotypes. The number of up-regulated / down-regulated DEGs is shown in the figure. C) Heat map representation of relevant Gene Ontology (GO) terms for differential expression based on bulk RNA sequencing.

[0036] Fig.14 : GFP and progerin counts based on RNA sequencing reads. The aging group shows a higher number of reads for GFP (upper figure) and progerin (lower figure), while the control non-aging group has no reads. However, for the Bob N2P system, the data show leakage from the cumate induction system, because the expression of GFP was detected in the iPSC (iPSC D0 N2P) not treated with cumate.

[0037] Fig.15 : A) Expression of iPSC marker genes in aged vs. non-aged iPSCs shows downregulation of Nanog and OCT4 (POU5F1). B) Volcano plot of iPSC marker genes in aged and non-aged iPSCs.

[0038] Fig.16 : Gene Ontology (GO) term analysis. The aged iNeuro group showed downregulation in GO-terms related to synaptic transmission, action potential, and morphogenesis, and upregulation in GO-terms related to DNA damage, apoptosis, and senescence at D14 and D21 after induction.

[0039] Fig.17 : Epigenetic clock analysis in aging iPSCs and iNeurons. A) Transcriptional clock analysis based on Tabula Muris Senis (Mouse Aging Cell Atlas). Both the "non-zero clock" and the "binarized" clocks were trained using Bulk RNA-seq data, indicating that epigenetic effects also exist in aging iNeurons. However, these clocks cannot predict iPSC age well because the data are compared with mouse brains. B) Epigenetic clock analysis is based on the Steve Horvath clock (Horvath 2013 Genome Biol). Although there seems to be a shift to an older age for the aging group, no significant differences were observed between non-aging and aging iPSCs / iNeurons. Bob N2P iPSCs treated with cumate showed similar results to those of untreated ones, supporting the possibility of leakage in the cumate-induced system. C) DNA methylation entropy analysis based on the number of methylated CpGs can predict aging well. Both Bob N2P iPSCs (left) and iNeurons (right) treated with cumate showed higher entropy than that generated by Bob NGNs, indicating epigenetic aging. **p<0.01, ***p<0.001. +cumate, 75μg / mL cumate; +cumate, 150μg / mL cumate.

[0040] Fig.18: Horvath epigenetic analysis shows a shift toward CD4 T cells. A) All aged cell lines (post-aging and pre-aging) show a heterogeneous population of neurons and non-neuronal-like cells after 14 days of culture. These non-neuronal cells did not appear until overexpression of progerin was triggered at D7. Scale bar = 100 μm. B) Horvath's epigenetic analysis shows that neurons lose their neuronal identity and shift toward CD4 T cells. 75 = 75 μg / mL cumate; 150 = 150 μg / mL cumate. C) Heatmaps of specific cell type markers (e.g., pluripotency, neurons, T cells) show no correlation between aged iNeurons and T cells.

[0041] Fig.19 . CRISPR screening to identify target genes involved in aging and regeneration. A) Transformation of Cas9 cell lines based on the progerin model. Expression of progerin (GFP) and Cas9 (mCherry) was observed (right). The Cas9KO efficiency was verified by the loss of B2M protein expression after delivery of specific sgRNA (red, unfilled) compared to the untreated control (gray, filled), thus confirming the Cas9 activity in this cell line. B) Schematic diagram of crop-sequencing screening based on single-cell RNA sequencing reads. C) The number of cells for each knockout identified. D) Linear discriminant analysis (LDA) using aging features was used to create an aging scale. The scale is integrated into the Uniform Manifold Approximation and Projection (UMAP) map of all single cells treated in the CRISPR knockout screen. E) Based on this aging scale, the EZH2 gene was identified as having anti-aging effects (left curve = EZH2 targeting, right curve = non-targeting control). DETAILED DESCRIPTION

[0042] According to a first aspect of the present invention, there is provided a method for transiently inducing aging in stem cells, the method comprising:

[0043] (i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0044] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0045] As used herein, the term "aging" and its various parts of speech refer to age-related phenotypic changes that limit functionality and can be considered as the result of aging. These aging phenotypes may be different from aging DNA-related factors such as epigenetic changes and double-strand breaks, the causal relationship between these factors and aging has not yet been determined, and the reversal of these factors may not lead to aging reversal or non-aging phenotype maintenance. However, it should be understood that these related factors can be measured as alternative / further aging phenotypes in the methods described herein. Examples of cell phenotypes associated with aging are: mitochondrial dysfunction, senescence, changes in intracellular communication, genomic instability, telomere shortening, epigenetic changes, and dysregulation of nutritional pathways (Otin et al. (2013) Cell "The Hallmarks of Aging"). Other aging phenotypes include: decreased proliferation, changes in cell and / or nuclear morphology, changes in gene expression, and upregulation of lamin A and / or lamin C nuclear proteins. Thus, "aging" and its various parts can refer to any aging phenotype herein, and in certain embodiments refers to the phenotype of a cell (e.g., an aging phenotype), such as a cellular phenotype measured as part of a method for identifying a gene or combination of genes described herein that is involved in reversing or maintaining a non-aging phenotype.

[0046] In one embodiment, the aging phenotype is selected from one or more of the following: proliferation, aging, cell and / or nuclear morphology changes, mitochondrial function, gene expression changes, upregulation of lamin A and / or lamin C nuclear proteins, epigenetic markers associated with aging, changes in methylation entropy, DNA double-strand breaks, telomere length, and transcriptome and / or epigenetic clocks. In another embodiment, the aging phenotype is a decrease in proliferation and / or an increase in senescence. In another embodiment, changes in nuclear morphology are abnormal folding, blistering, and / or loss of nuclear circularity. In yet another embodiment, mitochondrial function is reduced oxygen consumption and / or increased mitochondrial reactive oxygen species (ROS). In another embodiment, changes in gene expression are selected from one or more of the following: downregulation of somatic lineage identity genes, downregulation of mitochondrial genes, upregulation of apoptosis and / or aging-related genes, and upregulation of DNA damage-related genes. In another embodiment, epigenetic markers associated with aging are selected from one or more of the following: reduced heterochromatin trimethylation H3K9 (H3K9me3), reduced HP1γ, and increased γH2AX. In another embodiment, telomere length is shortened. In yet another embodiment, the transcriptome and / or epigenetic clock is a single cell transcriptome and / or epigenetic clock. In certain embodiments, the aging phenotype is a transcriptome clock, such as a transcriptome signature associated with an aging phenotype. In another embodiment, the aging phenotype is a single cell transcriptome clock, such as a transcriptome signature associated with an aging phenotype. In yet another embodiment, the aging phenotype, such as an aging phenotype of a cell, is a combination of one or more of the aging phenotypes described herein.

[0047] In one embodiment, an epigenetic clock is used, such as the Horvath epigenetic clock (Horvath, S (2013) Genome Biol., doi: https: / / doi.org / 10.1186 / gb-2013-14-10-r115) determines the "aging" of a cell or whether a cell is "aging". Therefore, in another embodiment, inducing cell aging includes aging the cell according to an epigenetic clock (e.g., the Horvath epigenetic clock). The Horvath epigenetic clock can be used as an age estimation method based on DNA methylation of CpG dinucleotide motifs in DNA. The DNA methylation age (further referred to as "predicted age") is characterized by the following properties: it is close to zero for ES cells and iPS cells; it is related to the number of cell passages; it produces a highly heritable accelerated measurement method for aging; and it is applicable to chimpanzee tissues. Studies have shown that the DNA methylation age of blood can predict all-cause mortality in late life even after adjusting for known risk factors, indicating that it is related to the process that leads to aging. Similarly, markers of physical and mental health are associated with epigenetic clocks. A particular feature of the Horvath epigenetic clock is its high accuracy and applicability to a wide spectrum of tissues and cell types. Since it allows the comparison of the age of different tissues and cells from the same subject (including forward programmed somatic cells with non-iPSC derived somatic cells of the same lineage or pluripotent cells such as iPSCs), it can be used to identify tissues and cells that show evidence of accelerated aging due to disease. In addition, the Horvath epigenetic clock can be used to identify any changes in DNA methylation age caused by treatments such as reprogramming or forward programming.

[0048] In another embodiment, "aging" is determined using a transcriptome clock, such as a single cell transcriptome clock. Thus, in another embodiment, inducing cellular aging comprises aging the cell according to a transcriptome clock. In some embodiments, the transcriptome clock comprises a transcriptome signature associated with an aging phenotype of the cell. Fleischer et al. (2018) Genome Biol. 19, 221 (doi: https: / / doi.org / 10.1186 / s13059-018-1599-6) describes an example of a transcriptome clock. Previously known aging clocks based on single-cell RNA sequencing feature training generally have the following problems: i) are limited to specific cell types; ii) have lower accuracy than epigenetic-based aging clocks; iii) have large differences in age measurements. However, these can be overcome in the methods described herein using a novel bioinformatics method to generate a single-cell transcriptome clock, which: i) is independent of cell type; ii) exhibits accuracy comparable to that of an epigenetic aging clock; and iii) has minimal differences in age measurements. The bioinformatics method can also be applied to multi-omics data, in which datasets such as genomic, proteomic, and epigenomic datasets are combined. In another embodiment, a transcriptome clock, such as a single-cell transcriptome clock, can be combined with any aging phenotype described herein. For example, in some embodiments, age is determined using a combination of a single-cell transcriptome clock with cell and / or nuclear morphology changes, epigenetic omics clock measurements, mitochondrial function, epigenetic marks associated with aging, changes in methylation entropy, gene expression, or any combination thereof.

[0049] Inducing aging according to the epigenetic and / or transcriptome clock as described herein includes inducing the age determined by the epigenetic and / or transcriptome clock to become "older" (i.e., such that a quantifiable change can be detected) when one or more aging-inducing factors are introduced into the cell or the cell is exposed to one or more exogenous aging-inducing factors, compared to the cell before the introduction or exposure to the aging-inducing factor (i.e., the methylation and / or transcriptome age is older than that of non-aged cells). Additionally or alternatively, inducing aging includes inducing the age determined by the epigenetic and / or transcriptome clock to become older compared to cells that have not introduced one or more aging-inducing factors or cells that have not been exposed to one or more exogenous aging-inducing factors.

[0050] In another embodiment, "aging" is determined using any suitable molecular features, including those suitable for use as biological clocks. Therefore, such biological clocks can be used to determine biological age. Other examples of biological clocks mentioned above are known in the art, including telomere length measurements, proteomic clocks, metabolomics clocks, and ribosomal clocks (rDNA; Wang & Lemos (2019) Genome Res, doi: http: / / www.genome.org / cgi / doi / 10.1101 / gr.241745.118) that measure the methylation state of CpG sites in ribosomal DNA. It is easy to understand that any of these and other molecular markers / biological clocks can be used independently or in combination (for example, epigenetic and transcriptomic clocks can be used in combination to determine age, or any one of epigenetic and transcriptomic clocks / both can be used together with another biological clock (such as a ribosomal clock)). Thus, in the case of a biological clock, making a cell "older" will include a detectable and quantifiable change in the age determined by these clocks, such as a more aged rDNA methylation (rDNAm) age, when one or more aging-inducing factors are introduced into the cell or the cell is exposed to one or more exogenous aging-inducing factors, compared to the cell before said introduction or exposure to the aging-inducing factors. Additionally or alternatively, inducing aging includes inducing the age determined by the biological clock (e.g., rDNAm age) to become older compared to a cell that has not been introduced with one or more aging-inducing factors or a cell that has not been exposed to one or more exogenous aging-inducing factors.

[0051] Therefore, the term "induced aging" used herein refers to the age change of the cells described herein, in particular to making the cells "older". Such changes in cell age include changes in the aging phenotype, epigenetic clock (i.e., methylation age) and / or transcriptome clock (i.e., transcriptome age) as described herein. Therefore, in some embodiments, inducing cell aging includes any one or more of the aging phenotypes described herein, for example, inducing one or more of the aging phenotypes described herein. In another embodiment, inducing cell aging includes changes in the epigenetic clock and / or transcriptome clock, in particular the transcriptome clock (i.e., transcriptome age). It should be understood that aging as described herein is imposed on cells by introducing or being exposed to one or more aging-inducing factors, and can therefore be referred to as "artificial". Therefore, in certain embodiments, compared with normal cell aging, for example, compared with cells of the same type undergoing the method, that is, compared with cell aging when artificial aging is not induced, the aging induced by the method of the present invention will be accelerated.

[0052] The term "induce ageing, inducing ageing, induced ageing, ageing-inducing" is used herein to indicate active induction of aging by the methods of the present invention. It should be understood that such induction may be temporary or transient, i.e., lasting for a defined period of time, rather than permanent. For example, the methods described herein induce aging in cells for a period of time (i.e., transiently) while introducing one or more aging inducing factors into cells or exogenously exposing them to cells, and then remove or reduce one or more aging inducing factors, thereby removing the induction of aging. Therefore, in one embodiment, aging is induced in stem cells or somatic cells for a period of time, followed by removal of one or more aging inducing factors. In a specific embodiment, removal or reduction of one or more aging inducing factors includes active removal or reduction, thereby resulting in the reversal of one or more aging phenotypes in the cell. Active removal or reduction of one or more aging inducing factors may include removal of exogenously provided aging inducing factors, inhibition of the activity of aging inducing factors, and / or reduction / inactivation of the expression of sequences encoding aging inducing factors. For example, where the introduction of one or more aging inducing factors includes one or more expression cassettes, the expression cassettes comprising sequences encoding one or more aging inducing factors, active removal or reduction may include reduction and / or inhibition of the expression of the sequences. It is easy to understand by the normal interpretation of the term "active", that such active removal or reduction does not include the natural turnover of aging-inducing factors introduced into cells, for example, by transient transfection. Therefore, in one embodiment, reducing and / or inhibiting the expression of sequences encoding one or more aging-inducing factors does not include passage of cells after transient transfection of sequences encoding one or more aging-inducing factors. Reducing and / or inhibiting the expression of sequences encoding one or more aging-inducing factors may include active regulation (i.e., reduction / inhibition) of the expression by a transcriptional regulatory protein, which binds to an inducible promoter and controls its expression. Therefore, in some embodiments, wherein the expression cassette also comprises an inducible promoter regulated by a transcriptional regulatory protein, the expression of sequences encoding one or more aging-inducing factors is reduced and / or inhibited by regulating the activity of the transcriptional regulatory protein, in particular, actively regulating the activity of the transcriptional regulatory protein. For example, when the transcriptional regulatory protein is a transcriptional activator protein, the expression of sequences encoding one or more aging-inducing factors is reduced / inhibited by removing exogenous substances (e.g., tetracycline and its derivatives or cumate) that activate the transcriptional activator protein from the cell. Alternatively, when the transcriptional regulatory protein is a transcriptional repressor protein, expression is reduced / inhibited by adding an exogenous substance that inactivates the transcriptional repressor protein (eg, tetracycline and its derivatives or cumate).Therefore, in another embodiment, the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is reduced and / or inactivated by removing exogenously provided substances, such as tetracycline and its derivatives or cumate. In another embodiment, one or more aging-inducing factors are introduced into cells or cells are exposed to one or more exogenous aging-inducing factors for a period of time, including introducing one or more expression cassettes into cells, wherein the expression cassettes comprise sequences encoding the one or more aging-inducing factors and an inducible promoter regulated by a transcriptional regulatory protein, wherein the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is controlled by exogenously provided substances such as tetracycline and its derivatives or cumate.

[0053] In step (i), aging is induced and one or more aging inducing factors are introduced into cells / exposed to the time period of cells, which should be understood to depend on the aging inducing factors and / or the amount of aging inducing factors introduced into cells or cells to which they are exposed. The identity of the stem cells or somatic cells that induce aging can also determine the time period for inducing aging. For example, proliferating stem cells or somatic cells can induce a shorter time than non-proliferating stem cells or somatic cells or stem cells or somatic cells that proliferate more slowly. Alternatively, stem cells with the regenerative ability to reverse the aging phenotype can induce a longer time than somatic cells that do not contain regenerative ability. Therefore, in some embodiments, aging is induced in step (i) for a period of time so that stem cells or somatic cells develop and / or display one or more aging phenotypes as described herein, such as until development and / or display aging phenotypes. In another embodiment, aging is induced in step (i) for a period of time so that one or more aging phenotypes as described herein are measured and / or observed in stem cells or somatic cells, such as until aging phenotypes are measured and / or observed. Therefore, in some embodiments, aging phenotypes are induced in step (i) for a period of time. In one embodiment, aging is induced in step (i) for 5 days or longer. Therefore, in another embodiment, one or more aging inducing factors are introduced into the cell, or the cell is exposed to one or more aging inducing factors for 5 days or longer. In another embodiment, aging is induced in step (i) for 7 days or less. Therefore, in one embodiment, one or more aging inducing factors are introduced into the cell, or the cell is exposed to one or more aging inducing factors for 7 days or less. In another embodiment, aging is induced for 5 to 7 days in step (i). Therefore, in one embodiment, one or more aging inducing factors are introduced into the cell, or the cell is exposed to one or more aging inducing factors for 5 to 7 days. In one embodiment, aging is induced for 5 days. Therefore, in one embodiment, one or more aging inducing factors are introduced into the cell, or the cell is exposed to one or more aging inducing factors for 5 days. In another embodiment, aging is induced for 7 days in step (i). Therefore, in one embodiment, one or more aging inducing factors are introduced into the cell, or the cell is exposed to one or more aging inducing factors for 7 days. "Inducing aging for a period of time" mentioned herein also applies to introducing one or more aging inducing factors or exposing cells to one or more exogenous aging inducing factors for a period of time in step (i). After removing or reducing one or more aging-inducing factors in step (ii), for example, after actively removing or reducing the aging-inducing factors as described above, the inventors observed that stem cells (e.g., pluripotent stem cells and iPSCs) reversed the changes in the aging phenotype seen when the aging-inducing factors were introduced, and that the reversal was faster than that explained by changes in the levels of the aging-inducing factors in the cells (e.g., by protein turnover).Therefore, without being bound by theory, it is assumed that the unique regenerative ability of stem cells actively reverses the aging phenotype of transient induction. Therefore, in certain embodiments, aging is induced in stem cells or somatic cells for a period of time in step (i), and then one or more aging induction factors are removed or reduced in step (ii), thereby causing the reversal of one or more aging phenotypes in cells. In a specific embodiment, aging is induced in stem cells or somatic cells for a period of time in step (i), and then one or more aging induction factors are actively removed or reduced in step (ii), thereby causing the reversal of one or more aging phenotypes in cells. In one embodiment, the one or more aging phenotypes induced in step (i) are the same aging phenotypes as the aging phenotypes reversed after removing or reducing the aging induction factors in step (ii). In an alternative embodiment, the one or more aging phenotypes induced in step (i) are aging phenotypes different from the aging phenotypes reversed in step (ii). In another embodiment, the one or more aging phenotypes induced in step (i) are the same as the one or more aging phenotypes reversed in step (ii).

[0054] Therefore, in another aspect of the present invention, a method for transiently inducing aging in stem cells is provided, the method comprising introducing one or more aging-inducing factors into cells, or exposing cells to one or more exogenous aging-inducing factors for a period of time, and then removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in cells. In certain embodiments, removing or reducing one or more aging-inducing factors is active removal or reduction as described above, resulting in the reversal of one or more aging phenotypes in cells.

[0055] The method described herein for transiently aging cells (e.g., stem cells or somatic cells derived from iPSCs by forward programming), which comprises a first step of introducing one or more aging-inducing factors into cells or exposing cells to the aging-inducing factors, followed by a second step of removing or reducing the aging-inducing factors, particularly actively removing or reducing the aging-inducing factors, has not been previously disclosed in the art. For example, US2016 / 0115444 discloses the ability to accelerate cell "maturation" using progerin, and Chojnowski et al. (2020) Aging Cell, 19(3):e13108 (doi: https: / / doi.org / 10.1111%2Facel.13108) discloses the effects of progerin expression on the cell cycle and heterochromatin loss in cells. However, both US2016 / 0115444 and Chojnowski et al. (2020) used transient expression of progerin in cells, and therefore neither discloses the reversal of aging in stem cells or iPSC-derived somatic cells after removing or reducing aging-inducing factors according to the present invention. In particular, there is no disclosure of actively removing or reducing one or more aging-inducing factors, thereby leading to the reversal of one or more aging phenotypes in cells. In addition, Miller et al. (2013) Cell Stem Cell, 13(6): 691-705 (doi: https: / / doi.org / 10.1016 / j.stem.2013.11.006 ) discloses the induction of various aging-related markers and features in fibroblasts and neurons following transient / short-term expression of progerin. However, there is no disclosure of removal / reduction of progerin or reversal of aging following transient expression of progerin, which may mean that transient expression of progerin is sufficient to irreversibly induce an aging phenotype, contrary to the inventors' findings described herein.

[0056] Therefore, the methods described herein can be used to screen for cellular processes and genes related to stem cell regeneration capacity, particularly genes and gene combinations related to aging phenotype reversal or non-aging phenotype maintenance (such as the phenotype of stem cells). Screening methods for identifying cellular processes or genes involved in stem cell regeneration capacity or aging phenotype reversal / non-aging phenotype maintenance have not been found in the art.

[0057] It should be understood that the term "aging-inducing factor" used herein can include any condition, agent, compound, gene / gene expression or cell condition that can induce and / or change any aging phenotype described herein, including the age determined using epigenetic and / or transcriptome clocks or other biological clocks / biological clock combinations. For example, the aging-inducing factor can be another splicing form of lamin A, such as presenilin or presenilin-like truncated form of presenilin A, which changes the nuclear morphology, particularly the loss of nuclear circularity. Presenilin is a lamin associated with Hutchinson-Gilford progeroid syndrome (HGPS), a mutant form of lamin A lacking 50 amino acids at the C-terminus, which prevents the removal of farnesyl and causes the accumulation of proteins at the nuclear edge / lamina. In another embodiment, the aging-inducing factor can be a physical stressor. Physical stressors include radiation, such as X-ray radiation and other inducers of DNA damage that cause DNA double-strand breaks. Therefore, in one embodiment, one or more aging-inducing factors are alternative splicing forms of lamin A. In a specific embodiment, the alternative splicing form of lamin A is presenilin. In another embodiment, one or more aging-inducing factors are presenilin-like proteins, such as truncated forms of lamin A, which may include a deletion of a C-terminal truncation site, resulting in incorrect processing of the protein, which cannot be integrated into the nuclear lamina, causing morphological changes in the cell, which are similar to those caused by presenilin. In another embodiment, the one or more aging-inducing factors are mitochondrial DNA mutations, such as deletions of DNA polymerase subunit γ (POLG). In another embodiment, the one or more aging-inducing factors are aging-inducing compounds, such as telomerase inhibitors (e.g., BIBR1532), reactive oxygen species (ROS), or compounds that interrupt mitochondrial function, such as 6-hydroxydopamine (6-OHDA), valinomycin, CCCP, rotenone, or hydrogen peroxide. In another embodiment, the aging-inducing factor is a physical stressor. In yet another embodiment, the aging-inducing factor is an inducer of DNA damage, such as ionizing radiation (e.g., X-ray radiation), a platinum-containing drug (e.g., cisplatin, oxaliplatin, or carboplatin), cyclophosphamide, chlorambucil, or temozolomide. In one embodiment, one or more aging-inducing factors are radiation, such as X-ray radiation.

[0058] In certain embodiments, the aging phenotype is specific to the cell, for example, specific to cell type and / or pedigree. For example, but not limited to, wherein the cell is a stem cell, such as a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a germ stem cell, a multipotent stem cell, an oligopotent stem cell, a unipotent stem cell or a tissue-specific / tissue-resident stem cell, the aging phenotype can be a proliferation reduction. Alternatively or in addition, when the cell is a stem cell, the aging phenotype can be an increase in senescence. If the cell is an immune cell, such as a T cell, the aging phenotype can be an increase in senescence. If the cell is a cell of the nervous system, such as a neuron or a glial cell, the aging phenotype can be a phenotype of a degenerative disease of the nervous system, such as a decrease in process density and / or connectivity, a shortening of the average dendrite length, a shortening of the neurite diameter, a reduction in neuronal marker genes or a decrease in electrophysiological activity. Therefore, in an embodiment in which the cell is a neuron, the aging phenotype is a phenotype of a degenerative disease of the nervous system, such as those described herein. If the cell is a myocyte, such as in the heart, the aging phenotype may be reduced electrophysiological activity or reduced mitochondrial function.

[0059] Therefore, in one embodiment, stem cells are pluripotent stem cells, induced pluripotent stem cells (iPSC), germ stem cells, multipotent stem cells, oligopotent stem cells, unipotent stem cells or tissue-specific / tissue-resident stem cells. Therefore, it should be understood that cells suitable for methods and uses described herein may include any type of stem cells. For example, stem cells may be pluripotent stem cells, such as iPSC, embryonic stem cells or pluripotent stem cells obtained by nuclear transfer or cell fusion. Preferably, embryonic stem cells are extracted without destroying the embryo, particularly when the cell is a human cell. In some embodiments, stem cells are not derived from human or animal embryos, i.e., the present invention does not extend to any method or use involving the destruction of human or animal embryos. Stem cells may also include pluripotent stem cells, oligopotent stem cells or unipotent stem cells. Stem cells may also include fetal stem cells or adult stem cells, such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells or skin stem cells. In some aspects, stem cells may be separated from umbilical cord, placenta, amniotic fluid, chorionic villi, blastocysts, bone marrow, adipose tissue, brain, peripheral blood, cord blood, menstrual blood, blood vessels, skeletal muscle, skin and liver.

[0060] "Pluripotent" mentioned herein refers to cells with the potential to differentiate into cells of all types / lineages found in an organism.Pluripotent stem cells can differentiate into a smaller number of cell types than pluripotent cells, such as only those closely related cell lineages.Oligopotent stem cells can only differentiate into a few cell types, such as lymphoid or myeloid stem cells.Unipotent cells can only produce a type of cell, and are therefore lineage-specific, but have the ability to self-renew, which distinguishes them from non-stem cells (e.g., progenitor cells that cannot self-renew).Of particular interest to the present invention is a form of pluripotent stem cells known as induced pluripotent stem cells." induced pluripotent stem cells" (iPSC) are cells that are reprogrammed to a similar embryonic stem cell state by forcing expression of genes and factors that are important for maintaining embryonic stem cell definition characteristics. In 2006, studies have shown that the overexpression of four specific transcription factors can convert adult cells into pluripotent stem cells.Some members of the Oct-3 / 4 and Sox gene families have been identified as potential key transcription regulators involved in the induction process. Other genes include some members of the Klf family, the Myc family, Nanog and Lin28, which may improve the induction efficiency. Examples of genes that can be used as reprogramming factors to produce iPSC include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Lin28b, Sall4, Esrrb, Tbx3 and Glis1, GATA3, GATA6, and these reprogramming factors can be used alone or in combination of two or more thereof. In particular, the reprogramming factor can include at least Yamanaka factors, i.e. Oct3 / 4, Sox2, Klf4 and c-Myc.

[0061] In an alternative embodiment, the cell is a somatic cell, such as a somatic cell derived from a reprogrammed iPSC. The iPSC may be as defined herein.

[0062] Therefore, according to another aspect of the present invention, there is provided a method for transiently inducing aging in somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising:

[0063] (i) before or after forward programming of iPSCs, introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0064] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0065] On the other hand, a method for transiently inducing aging in somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming is provided, the method comprising introducing one or more aging-inducing factors into cells before or after the forward programming of iPSCs, or exposing cells to one or more exogenous aging-inducing factors for a period of time, and then removing or reducing one or more aging-inducing factors, thereby causing the reversal of one or more aging phenotypes in cells. In certain embodiments, removing or reducing one or more aging-inducing factors is active removal or reduction as described above, resulting in the reversal of one or more aging phenotypes in cells.

[0066] In one embodiment, the somatic cell is a neuron (e.g., a motor neuron, a sensory neuron, a GABAergic neuron, a glutamatergic neuron, or a dopaminergic neuron), a glial cell, a blood cell (e.g., an erythrocyte), an immune cell (e.g., a T cell, a B cell, a macrophage, a NK cell, a neutrophil, or a granulocyte), a liver cell (e.g., a hepatocyte, a Kupffer cell, or a stromal cell), a muscle cell (e.g., a smooth muscle cell), a muscle cell (e.g., a cardiomyocyte), a fibroblast, a skin cell (e.g., a keratinocyte), a bone cell, a chondrocyte, an epithelial cell, an endothelial cell, or an adipocyte. In another embodiment, the cell, e.g., a somatic cell, is a neuron derived from iPSC by forward programming.

[0067] The term "somatic cell" as used herein includes cells of any type (i.e., pedigree) constituting an organism, excluding germ cells and undifferentiated stem cells. Therefore, somatic cells may include, for example, but not limited to, neurons, glial cells, blood cells (e.g., leukocytes), liver cells (e.g., hepatocytes (hepatocytes)), muscle cells (e.g., myocytes), or fibroblasts. In one embodiment, somatic cells may be adult cells or cells from adults, which exhibit one or more detectable features of adult or non-embryonic cells. The somatic cells "derived from aniPSC by forward programming" mentioned herein refer to such cells, which contain the phenotype and / or features of somatic cells as defined herein (e.g., surface phenotypes and / or functional features associated with specific pedigrees) and have been forward programmed from pluripotent stem cells that have been previously reprogrammed as described herein, i.e., iPSC. The forward programming of iPSC to somatic cells includes the introduction of pedigree-specific factors, such as transcription factors, or nucleic acids encoding the pedigree-specific factors, such as in the form of mRNA or expression cassettes. Alternatively, forward programming may include increasing the expression of a lineage-specific factor (e.g., a lineage-specific transcription factor), such as by increasing the expression of the lineage-specific factor gene and / or its protein expression. The expression of an exogenous or endogenous (particularly exogenous) transcription factor may be increased. In one embodiment, forward programming includes introducing into iPSC a nucleic acid or protein product encoding or providing a lineage-specific transcription factor or a combination thereof, and culturing cells under conditions suitable for reprogramming cells into somatic cells.

[0068] It should be understood that increasing the expression of lineage-specific factors (e.g., transcription factors) in iPSCs to be forward programmed as somatic cells may include any method known in the art, for example, by inducing the expression of one or more expression cassettes previously introduced into the cell, or by introducing nucleic acids (e.g., DNA or RNA), polypeptides, or small molecules into the cell. Increasing the expression of certain endogenous but transcriptionally inhibited genes may also reverse the silencing or inhibitory effects on the expression of these genes by regulating upstream transcription factor expression or epigenetic regulation. Therefore, the method of the present invention may include culturing a cell population under certain conditions to artificially increase the expression level of one or more lineage-specific transcription factors.

[0069] It should also be understood that such methods of increasing expression, such as by inducing expression of one or more expression cassettes or by introducing nucleic acids, polypeptides or small molecules described herein, can also be used to introduce one or more aging-inducing factors into cells or expose cells to one or more exogenous aging-inducing factors, and to change the expression and / or activity of one or more genes or gene combinations identified as involved in the reversal of an aging phenotype or the maintenance of an unaged phenotype in cells by the methods described herein. Therefore, the methods of increasing the expression of lineage-specific transcription factors mentioned herein are also applicable to increasing the expression / introduction of the one or more aging-inducing factors, and vice versa, and to changing the expression / activity of one or more genes or gene combinations identified by the methods described herein. Related methods of controlling expression can also be used to actively remove / reduce the one or more aging-inducing factors by reducing / suppressing the expression of sequences encoding the one or more aging-inducing factors.

[0070] Therefore, in one embodiment, the expression of lineage-specific transcription factors is increased by contacting iPSC to be forward programmed with transcription factors (i.e., transcription factor proteins). In another embodiment, one or more aging-inducing factors are introduced by contacting stem cells or somatic cells with the aging-inducing factors. Lineage-specific transcription factor proteins and / or aging-inducing factors can be delivered to cells by direct electroporation. Alternatively, aging-inducing factors can be delivered by direct treatment or application to cells, for example, wherein aging-inducing factors are aging-inducing compounds, and / or by exposing cells to physical stressors, such as radiation. As understood by the methods described herein, aging-inducing factors can be introduced before the expression of lineage-specific transcription factors is improved, or can be introduced after the expression of lineage-specific transcription factors is improved, i.e., before or after the forward programming of iPSC is introduced. Therefore, in one embodiment, one or more aging-inducing factors are first introduced into iPSC, and then iPSC is contacted with lineage-specific transcription factors. In an alternative embodiment, iPSC is first contacted with lineage-specific transcription factors, and then one or more aging-inducing factors are introduced into iPSC.

[0071] In an alternative embodiment, the expression of lineage-specific transcription factors is increased by contacting the iPSC to be forward programmed with one or more activating or increasing the expression or quantity of the transcription factor. In another embodiment, one or more aging-inducing factors are introduced by contacting stem cells and / or somatic cells with one or more activating or increasing the expression of the aging-inducing factor. The expression of aging-inducing factors can be activated or increased before the expression of lineage-specific transcription factors increases, or can be activated or increased after the expression of lineage-specific transcription factors increases, i.e., activated or increased before or after the forward programming of iPSC, respectively. Therefore, in one embodiment, iPSC is first contacted with one or more activating or increasing agents for the expression of one or more aging-inducing factors, and then iPSC is contacted with one or more agents for increasing the expression of lineage-specific transcription factors. In an alternative embodiment, iPSC is first contacted with one or more agents for increasing the expression of lineage-specific transcription factors, and then iPSC is contacted with one or more agents for activating or increasing the expression of one or more aging-inducing factors.

[0072] In one embodiment, the agent is selected from: nucleic acids (i.e., polynucleotides, such as messenger RNA (mRNA) or coding DNA sequences); proteins (e.g., antibodies); aptamers and small molecules; ribosomes; RNAi agents; guide RNA (gRNA); and peptide nucleic acids (PNA) and their analogs or variants. In one embodiment, the agent is a transcriptional activation system (e.g., a gRNA for a gene activation system such as CRISPR / Cas9 or TALEN) for increasing the expression of one or more endogenous lineage-specific transcription factors and / or aging-inducing factors.

[0073] Forward programming & introduction / removal of aging-inducing factors

[0074] The method of forward programming iPSC to somatic cells can include delivering nucleic acid to cells, the nucleic acid comprising one or more open reading frames of a transcriptional activator encoding a lineage-specific transcription factor (e.g., in an expression cassette), a transcription factor protein, or an open reading frame encoding the transcription factor. This results in an increase in the amount of lineage-specific transcription factors in cells, and cells are programmed to form somatic cells. The open reading frame can be a part of a recombinant expression cassette. The introduction of one or more aging-inducing factors can also include delivering nucleic acid to cells, the nucleic acid comprising an open reading frame of a transcriptional activator encoding the aging-inducing factor or an open reading frame encoding the aging-inducing factor.

[0075] In one embodiment, nucleic acid comprises a recombinant or exogenous expression cassette, and the recombinant or exogenous expression cassette comprises a sufficient number of one or more lineage-specific transcription factor sequences (or genes) to cause iPSC to be forward programmed as somatic cells. In another embodiment, nucleic acid comprises a recombinant or exogenous expression cassette, and the recombinant or exogenous expression cassette comprises a sufficient number of one or more aging-inducing factor sequences (e.g., sequences encoding aging-inducing factors) to induce the aging of cells as described herein, for example, by inducing an aging phenotype. The exogenous expression cassette may include an external inducible transcriptional regulatory element for the inducible expression of one or more transcription factors and / or aging-inducing factors, such as an inducible promoter, for example, comprising a tetracycline response element or a variant thereof. In another embodiment, nucleic acid comprises a recombinant or exogenous expression cassette, and the recombinant or exogenous expression cassette comprises one or more lineage-specific transcription factors and one or more aging-inducing factors (e.g., sequences encoding transcription and aging-inducing factors), thereby inducing iPSC to be forward programmed as somatic cells and inducing cell aging. Therefore, the exogenous expression cassette may contain two or more external inducible transcriptional regulatory elements for the inducible expression of one or more transcription factors and aging-induced factors, such as two or more different inducible transcriptional regulatory elements (e.g., inducible promoters) for the simultaneous or independent inducible expression of one or more transcription factors and aging-induced factors.

[0076] If the expression of lineage-specific transcription factors and / or one or more aging-inducing factors is increased by introducing exogenous sequences encoding transcription and / or aging-inducing factors (e.g., transcription factor genes and / or aging-inducing factor genes), it should be understood that any suitable system can be used to deliver the sequence. The gene delivery system can be a transposon system; a viral gene delivery system; an episomal gene delivery system; or a homologous recombination system, such as using zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or meganucleases, or CRISPR / Cas9, etc.

[0077] Alternatively, as described herein or known to those of ordinary skill in the art, nucleic acid such as DNA or RNA is introduced into cells and any suitable nucleic acid delivery method can be used to transform cells. These methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection, by injection (including microinjection), by electroporation, by calcium phosphate precipitation, by using DEAE-dextran followed by polyethylene glycol, by direct sonic loading, by liposome-mediated transfection, by receptor-mediated transfection, by microparticle bombardment, by stirring with silicon carbide fibers, by Agrobacterium-mediated transformation and any combination of these methods. By applying these techniques, cells can be stably or transiently transformed. For example, transient transfection of nucleic acids encoding one or more aging-inducing factors can be used in the method of the present invention, for introducing one or more aging-inducing factors into cells or exposing cells to one or more exogenous aging-inducing factors for a period of time in step (i). Therefore, step (i) may include transient transfection. In a certain embodiment, wherein the cell is a stem cell, step (i) includes transient transfection. In addition, one or more expression cassettes can be transiently transfected into cells. Therefore, in another embodiment, one or more expression cassettes are transiently transfected into stem cells. Further, removing or reducing one or more aging-inducing factors in step (ii) can also include transient transfection, because such transfection introduces one or more aging-inducing factors into cells for a period of time, after which the nucleic acid encoding one or more aging-inducing factors is cleared by the cell, no longer expressed in the cell, or its expression is reduced in the cell. Therefore, step (ii) can include culturing cells, that is, effectively reducing or removing one or more aging-inducing factors in the cells. In one embodiment, step (ii) includes culturing stem cells, that is, effectively reducing or removing one or more aging-inducing factors in the stem cells. Additionally and / or alternatively, step (ii) can include passage cells in culture. Therefore, in another embodiment, step (ii) includes passage stem cells in culture. However, as understood by the disclosure herein, wherein the introduction / expression of one or more aging-inducing factors is carried out by controlled expression of sequences encoding the aging-inducing factors (e.g., using exogenously provided substances), within a period of time in step (i), transient transfection of nucleic acids encoding one or more aging-inducing factors is not performed to introduce the aging-inducing factors. Thus, in certain embodiments, step (i) does not include transient transfection. In a specific embodiment, wherein the cell is a somatic cell derived from iPSC by forward programming, step (i) does not include transient transfection. In another embodiment, the one or more expression cassettes are not transiently transfected into the cell, i.e., it is stably introduced into the cell as described herein and / or using methods known in the art.In another embodiment, the one or more expression cassettes are not transiently transfected into somatic cells derived from forward-programmed iPSCs. According to these embodiments, in step (ii), removing or reducing one or more aging-inducing factors also does not include transient transfection. For example, removing or reducing one or more aging-inducing factors is active removal / reduction, which does not rely on the removal of nucleic acids by cells. In one embodiment, step (ii) does not include passage of cells in culture. In another embodiment, step (ii) does not include passage of somatic cells derived from forward-programmed iPSCs in culture.

[0078] In addition, expression cassettes (e.g., inducible recombinant expression cassettes comprising sequences encoding lineage-specific transcription factors and / or one or more aging-inducing factors) may include cleavable sequences. These sequences are sequences that can be recognized by entities that can specifically cut DNA, including restriction sites, which are target sequences of restriction enzymes or sequences recognized by other DNA cutting entities, such as nucleases, recombinases, ribozymes or artificial constructs. At least one cleavable sequence may be included, but preferably there are two or more cleavable sequences. These cleavable sequences may be located at any suitable point in the cassette, so that if necessary, the selected portion or the entire cassette may be selectively removed. Therefore, cleavable sites may be located at the flanks of the partial / whole gene sequences that may need to be removed. Therefore, the method may also include removing expression cassettes and / or genetic material.

[0079] Carrier

[0080] In one embodiment, a vector is used to introduce lineage-specific transcription factors and / or one or more aging-inducing factors into a cell population. One skilled in the art can construct vectors by standard recombinant techniques. Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).

[0081] In one embodiment, the vector is a viral vector. The viral gene delivery system can be a viral vector based on RNA or based on DNA. The viral vector includes a retroviral vector, a slow viral vector (e.g., derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), a gamma retroviral vector, an adenovirus (Ad) vector (including its strong replication ability, replication defect and gutless form), an adeno-associated virus derived (AAV) vector, a simian virus 40 (SV-40) vector, a bovine papilloma virus vector, an Epstein-Barr virus vector, a herpes virus vector, a vaccinia virus vector, a Harvey mouse sarcoma virus vector, a mouse mammary tumor virus vector, a Rous sarcoma virus vector and a Sendai virus vector. In another embodiment, the viral vector is selected from: a slow viral vector, an adeno-associated virus vector or a Sendai virus vector. In another embodiment, the viral vector is a slow viral vector.

[0082] Lentiviral vectors are well known in the art. Lentiviral vectors are complex retroviruses that can be randomly integrated into the host cell genome, which, in addition to the common retroviral genes gag, pol and env, also contain other genes with regulatory or structural functions (e.g., accessory genes Vif, Nef, Vpu, Vpr). Lentiviral vectors have the advantage of being able to infect undivided cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, a recombinant lentiviral vector capable of infecting non-dividing cells, wherein a suitable host cell is transfected by two or more vectors carrying packaging functions, i.e., gag, pol and env, as well as rev and tat.

[0083] In one embodiment, nucleic acid sequences encoding lineage-specific transcription factors and / or one or more aging-inducing factors are introduced into the cell via a plasmid. In one embodiment, at least one nucleic acid sequence encoding a lineage-specific transcription factor is introduced into the cell on a single plasmid. In another embodiment, at least one nucleic acid sequence encoding a lineage-specific transcription factor and one or more aging-inducing factors is introduced into the cell on a single plasmid. In an alternative embodiment, sequences encoding lineage-specific transcription factors and one or more aging-inducing factors are introduced into the cell via different plasmids.

[0084] In one embodiment, plasmid is episomal. Episomal vectors can introduce large fragment DNA into cells, but remain outside chromosomes, replicate once per cell cycle, are effectively distributed to daughter cells, and do not cause immune response substantially. In alternative embodiments, episomal vectors based on Epstein-Barr virus (EBV), vectors based on yeast, vectors based on adenovirus, episomal vectors based on simian virus 40 (SV40), or vectors based on bovine papilloma virus (BPV) can be used.

[0085] Site-specific delivery and targeting

[0086] Any suitable technique can be used to insert the nucleic acid sequence into a specific sequence, and several techniques are described in the art. Suitable techniques include any method that introduces a break at the desired position and allows the vector to be recombined into the gap. Therefore, the key first step in target site-specific genome modification is to produce a double-stranded DNA break (DSB) at the genomic locus to be modified. Different cell repair mechanisms can be used to repair DSB and introduce the desired sequence, including non-homologous end joining repair (NHEJ) that is more prone to error; and homologous recombination repair (HR) mediated by a donor DNA template, which can be used to insert an inducible box.

[0087] There are several technologies to allow customized site-specific generation of DSBs in genomes. Many of these technologies involve the use of customized endonucleases, such as zinc finger nucleases, TALENs, or clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins (CRISPR / Cas9) systems.

[0088] Zinc finger nucleases are artificial enzymes produced by fusing zinc finger DNA binding domains to the nuclease domain of the restriction enzyme FokI. The latter has a non-specific cleavage domain that must dimerize to cut DNA. This means that two zinc finger nuclease monomers are required to allow dimerization of the FokI domain and cut DNA. The DNA binding domain can be designed to target any genomic sequence of interest and is a tandem array of Cys2His2 zinc fingers, each zinc finger recognizing three consecutive nucleotides in the target sequence. The two binding sites are 5-7bp apart to allow optimal dimerization of the FokI domain. Therefore, the enzyme is able to cut DNA at a specific site and increase target specificity by ensuring that two adjacent DNA binding events must occur to achieve double-strand breaks.

[0089] Transcription activator-like effector nucleases or TALENs are dimeric transcription factors / nucleases. They are made by fusing the TAL effector DNA binding domain to a DNA cleavage domain (nuclease). Transcription activator-like effectors (TALEs) can be engineered to bind virtually any desired DNA sequence, so when combined with a nuclease, the DNA can be cleaved at a specific location. TAL effectors are proteins secreted by bacteria of the genus Xanthomonas, whose DNA binding domains contain a repeated, highly conserved sequence of 33–34 amino acids, in which the 12th and 13th amino acids differ. These two positions are highly variable and show a strong correlation with specific nucleotide recognition. This direct relationship between amino acid sequence and DNA recognition allows the design of specific DNA binding domains by selecting a combination of repeated segments containing appropriate residues at the two variable positions. TALENs are thus constructed from an array of 33 to 35 amino acid modules, each targeting a single nucleotide. By selecting an array of modules, almost any sequence can be targeted. Likewise, the nuclease used can be FokI or a derivative thereof.

[0090] Three CRISPR mechanisms have been identified, of which type II is the most studied. The CRISPR / Cas9 system (type II) uses the Cas9 nuclease to produce double-strand breaks in DNA at sites determined by short guide RNAs. The CRISPR / Cas system is a prokaryotic immune system that confers resistance to exogenous genetic elements. CRISPR is a prokaryotic DNA fragment containing a short repeated base sequence. Each repetition is followed by a short fragment "protospacer DNA" from a previous contact with an exogenous genetic element. The CRISPR spacer uses RNA interference to recognize and cut exogenous genetic elements. The CRISPR immune response occurs in two steps: CRISPR-RNA (crRNA) biogenesis and crRNA-guided interference. CrRNA molecules consist of variable sequences transcribed from protospacer DNA and CRISPR repeat sequences. Then, each crRNA molecule hybridizes with a second RNA called trans-activating CRISPR RNA (tracrRNA), and the two eventually form a complex with the nuclease Cas9. If the complementary target DNA sequence is adjacent to a short sequence known as a protospacer adjacent motif (PAM), the portion of the protospacer DNA encoding of crRNA guides Cas9 to cut the complementary target DNA sequence. In many other applications, this natural system has been designed and utilized to introduce DSB breaks in a specific site of genomic DNA. In particular, the CRISPR II type system from Streptococcus pyogenes can be used. Most simply, the CRISPR / Cas9 system includes two components delivered to cells to provide genome editing: Cas9 nuclease itself and guide RNA (gRNA). GRNA is a fusion of customized site-specific crRNA (for target sequence) and standardized tracrRNA.

[0091] Once the DSB is made, a donor template with homology to the target locus is provided; the DSB can be repaired via the homology-directed repair (HDR) pathway, allowing for precise insertion.

[0092] Derivatives of this system are also possible. There are mutant forms of Cas9, such as Cas9D10A, which only have nickase activity. This means that it only cuts one DNA strand and does not activate NHEJ. Instead, when a homologous repair template is provided, DNA repair is performed exclusively through the high-fidelity HDR pathway. Cas9D10A can be used in paired Cas9 complexes designed to generate adjacent DNA nicks in conjunction with two sgRNAs complementary to adjacent regions on opposite strands of the target site, which may be particularly advantageous.

[0093] Elements for making double-stranded DNA breaks can be introduced into one or more vectors, such as plasmids, for expression in stem cells or somatic cells as described herein.

[0094] Thus, in the methods of the present invention, any method for making specific, targeted double-strand breaks in the genome to effect gene / inducible cassette insertion may be used. Preferably, the method for inserting a gene / inducible cassette utilizes any one or more of zinc finger nucleases, TALENs and / or CRISPR / Cas9 systems or any derivatives thereof.

[0095] Once the DSB is prepared by any suitable method, the gene / inducible cassette for insertion can be provided in any suitable manner as described herein. The gene / inducible cassette and associated genetic material form the donor DNA for repairing the DNA at the DSB and are inserted using standard cellular repair mechanisms / pathways. As described above, how the break is initiated will change which pathway is used to repair the damage.

[0096] Controlled expression

[0097] In one embodiment, the expression of lineage-specific transcription factors is under controlled transcription. In this aspect of the invention, the transcription and translation (expression) of transcription factors can be controlled in cells. If necessary, this allows overexpression of transcription factors. In another embodiment, the expression of the one or more aging-inducing factors is under controlled transcription. Therefore, in certain embodiments, one or more aging-inducing factors are introduced into cells using one or more expression cassettes comprising sequences encoding one or more aging-inducing factors, that is, one or more expression cassettes comprising sequences encoding one or more aging-inducing factors are introduced into cells in step (i).

[0098] The exogenous expression cassette carrying lineage-specific transcription factors and / or one or more aging-inducing factors may include an external inducible transcriptional regulatory element (i.e., an inducible promoter) for inducing transcription and / or expression of aging-inducing factors. The inducible expression cassette may be controlled by adding exogenous substances. Regardless of the culture conditions used, the exogenous substances will control the expression of the genetic sequence in the inducible expression cassette; and may be supplied continuously and then withdrawn to induce transcription, or supplied when transcription is required according to its mode of action.

[0099] The expression of lineage-specific transcription factors and / or one or more aging-inducing factors described herein can be increased using the dual-box expression system described in WO2018096343, which is incorporated herein by reference. The system targets genetic safe harbor (GSH) sites, which reduces the risk of epigenetic silencing of inserted genetic material.

[0100] Thus, in one embodiment, a sequence encoding a lineage-specific transcription factor and / or one or more aging-inducing factors as described herein is introduced into a stem cell or somatic cell using a method comprising:

[0101] - targeted insertion of a gene encoding a transcriptional regulatory protein into a first genetic safe harbor site of the cell; and

[0102] - Targeted insertion of an inducible cassette into a second genetic safe harbor site of the cell, wherein the inducible cassette comprises the transcription factor and / or one or more aging-inducible factor sequences operably linked to an inducible promoter, and the promoter is regulated by the transcriptional regulatory protein.

[0103] These embodiments of the invention provide a dual expression cassette system. Insertion of a gene encoding a transcriptional regulatory protein into a first genetic safe harbor (GSH) provides a control mechanism for the expression of an inducible cassette, which is operably linked to an inducible promoter and inserted into a second GSH site. In one embodiment, the first GSH and the second GSH are different.

[0104] In a specific embodiment, a gene encoding a transcriptional regulatory protein is inserted into a first gene safe harbor site, and an expression cassette comprising a sequence encoding one or more aging-inducing factors is inserted into a second gene safe harbor site.

[0105] wherein the expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein, and

[0106] The first genetic safe harbor site and the second genetic safe harbor site are different.

[0107] In an alternative embodiment, the expression cassette and the gene encoding the transcriptional regulatory protein are inserted into a genetic safe harbor site, such as a single genetic safe harbor site.

[0108] The expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein.

[0109] A GSH site is a locus within the genome where a gene or other genetic material can be inserted without any deleterious effects on the cell or the inserted genetic material. The most beneficial is a GSH site where the expression of the inserted gene sequence is not interfered with by any read-through expression from neighboring genes, and the expression of the inducible cassette minimizes interference with the endogenous transcriptional program. More formal criteria have been proposed that help determine whether a particular locus will be a GSH site in the future (Papapetrou et al. (2011)). These criteria include sites that are: (i) 50 kb or more from the 5' end of any gene; (ii) 300 kb or more from any gene associated with cancer; (iii) 300 kb or more from any microRNA (miRNA); (iv) located outside the transcription unit; and (v) located outside the ultra-conserved region (UCR). It may not be necessary to meet all of these proposed criteria, as the GSHs that have been identified do not meet all of them. It is believed that a suitable GSH will meet at least 2, 3, 4, or all of these criteria. Any suitable GSH site can be used in the methods of the present invention, provided that the site allows the insertion of genetic material without deleterious effects on the cell and allows transcription of the inserted genetic material. Those skilled in the art can use these simplified criteria to identify suitable GSHs and / or the more formal criteria described above.

[0110] In one embodiment, the first gene safe harbor site (GSH) and the second gene safe harbor site are selected from the hROSA26 locus, the AAVS1 locus, the CLYBL gene, the CCR5 gene or the HPRT gene (particularly any two thereof). Insertion in a gene safe harbor site is particularly preferred over random genomic integration because it is expected to be a safer modification of the genome and is less likely to cause adverse side effects, such as silencing native gene expression or causing mutations that lead to cancer cell types. In another embodiment, a single gene safe harbor site is selected from any GSH described herein.

[0111] The adeno-associated virus integration site 1 locus (AAVS1) is located within the protein phosphatase 1 regulatory subunit 12C (PPP1R12C) gene on human chromosome 19, which is uniformly and ubiquitously expressed in human tissues. AAVS1 has been shown to be a favorable environment for transcription because it contains an open chromatin structure and a natural chromosomal insulator that renders the inducible cassette resistant to silencing. It is not yet clear what adverse effects the disruption of the PPP1R12C gene has on cells. Furthermore, inducible cassettes inserted into this site remain transcriptionally active in many different cell types.

[0112] Based on sequence similarity to mouse GSH, the hROSA26 locus (ROSA26-backsplicing acceptor site #26) has been identified. The hROSA26 locus is located on chromosome 3 (3p25.3) and can be found in the Ensembl database (GenBank: CR624523). The integration site is located within the open reading frame (ORF) of the THUMPD3 long noncoding RNA (reverse strand). Since the hROSA26 locus has an endogenous promoter, the inserted genetic material can utilize the endogenous promoter, or alternatively, can be inserted in an operably linked manner to the promoter.

[0113] Intron 2 of the citrate lyase beta-like (CLYBL) gene on the long arm of chromosome 13 was identified as a suitable GSH because it is one of the identified integration hotspots for the phage-derived phiC31 integrase. Studies have demonstrated that inducible cassettes randomly inserted into this locus are stable and expressible. Insertion of inducible cassettes at this GSH has been shown not to interfere with local gene expression (Cerbini et al. (2015)). CLYBL therefore provides a GSH that may be suitable for use in the present invention.

[0114] CCR5, located on chromosome 3 (position 3p21.31), is the gene encoding the major co-receptor for HIV1. Interest in using this site as a GSH arises from null mutations in this gene that appear to have no adverse effects but tend to confer resistance to HIV-1 infection. Zinc finger nucleases have been developed that target the third exon, thus allowing insertion of genetic material at this locus.

[0115] The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene encodes a transferase that plays a key role in the generation of purine nucleotides via the purine salvage pathway.

[0116] GSHs have been identified in other organisms and include the ROSA26, HRPT, and Hipp11 (H11) loci in mice. Mammalian genomes may include GSH loci based on pseudo-attP sites. For these sites, the Streptomyces phage-derived recombinase hiC31 integrase has been developed as a non-viral insertion tool because of its ability to integrate plasmids containing inducible cassettes carrying attB sites into pseudo-attP sites.

[0117] Technically, the insertion of the first and / or second GSH can occur on one chromosome, or on two chromosomes. The GSH is present in the same locus on two chromosomes of a diploid organism. Insertion in two chromosomes is advantageous because it can increase the transcription level of the inserted genetic material from the inducible cassette, thus achieving particularly high levels of transcription.

[0118] DNA double-strand breaks can be generated based on customized site-specificity at the GSH, and genetic material can be specifically inserted into a specific GSH. The genetic material can then be introduced using any suitable mechanism, such as homologous recombination. Any method that generates a specific DSB in the genome can be used, but preferred systems include CRISPR / Cas9 and its modified forms, zinc finger nucleases, and TALEN systems.

[0119] One or more gene sequences can be controllably transcribed from the second and / or another GSH or from a single GSH. In fact, the inducible box can contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 gene sequences (e.g., aging induction factor sequences), it is desirable to insert them into the GSH, and controllably induce their transcription. Therefore, the lineage-specific transcription factor and / or one or more aging induction factors can be included in the same box that introduces the second gene safe harbor site. For example, the lineage-specific transcription factor and / or one or more aging induction factors can be included in several monocistronic constructs or one or more bicistronic or tricistronic constructs as required. It should be understood that similar combinations of constructs can be used to achieve higher expression. In one embodiment, the lineage-specific transcription factor and one or more aging induction factors are included in a bicistronic construct.

[0120] Alternatively, in the case of using a sequence encoding a lineage-specific transcription factor and one or more aging-inducing factors, the sequence encoding the lineage-specific transcription factor can be introduced into a separate GSH and / or controlled by different inducible promoters of the sequence encoding one or more aging-inducing factors. Therefore, in one embodiment, the lineage-specific transcription factor is introduced into a GSH separated from the aging-inducing factor. This can be achieved by utilizing two or more different GSH sites or utilizing the fact that GSH is present in the same locus on two chromosomes of a diploid organism, such as introducing a transcription factor into a GSH on a chromosome, and introducing one or more aging-inducing factors into the same GSH on another chromosome. If different expression levels or expression times of transcription factors and aging-inducing factors are required, this embodiment is advantageous. In an alternative embodiment, the sequence encoding the lineage-specific transcription factor and one or more aging-inducing factors is introduced into the same GSH, but controlled by different inducible promoters. Different expression levels or expression times of transcription factors and aging-inducing factors are also achieved using different inducible promoters. For example, the use of different inducible promoters allows the introduction of aging-inducing factors into somatic cells derived from forward-programmed iPSCs before the forward programming (i.e., where the expression of the aging-inducing factors has been induced before the expression of the lineage-specific transcription factors) or after the forward programming (i.e., where the expression of the aging-inducing factors is induced after the expression of the lineage-specific transcription factors). Therefore, in certain embodiments, wherein the cells are somatic cells derived from iPSCs by forward programming, a sequence encoding one or more aging-inducing factors is introduced into the same GSH as a sequence encoding a lineage-specific transcription factor, wherein each aging-inducing factor coding sequence and lineage-specific transcription factor sequence are controlled by different inducible promoters.

[0121] Transcriptional regulatory proteins are proteins that bind to DNA, preferably sequence-specifically to a DNA site located in or near a promoter, and promote the binding of the transcription machinery to the promoter, thereby promoting transcription of the DNA sequence (transcription activators) or blocking the process (transcription repressors).

[0122] The DNA sequences to which transcriptional regulatory proteins bind are called transcription factor binding sites or response elements, and these are present in or near the promoter of the regulated DNA sequence. Transcriptional activator proteins bind to response elements and promote gene expression. In the methods of the present invention, these proteins are preferably used to control inducible expression cassettes. Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0123] Transcriptional regulatory proteins can be activated or inactivated by many mechanisms, including binding of substances, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or co-regulatory proteins, phosphorylation and / or methylation. Transcriptional regulatory proteins can be controlled by activation or inactivation.

[0124] If the transcriptional regulatory protein is a transcriptional activator protein, then preferably the transcriptional activator protein needs to be activated. This activation can be performed by any suitable means, but preferably, the transcriptional regulatory protein is activated by adding an exogenous substance to the cell. The supply of exogenous substances to the cell can be controlled, so that the activation of the transcriptional regulatory protein can be controlled. Alternatively, an exogenous substance can be provided to inactivate the transcriptional regulatory protein, and then the recovered exogenous substance is provided to activate the transcriptional regulatory protein.

[0125] If the transcriptional regulatory protein is a transcriptional repressor protein, preferably the transcriptional repressor protein needs to be inactivated. Therefore, a substance is provided to prevent the transcriptional repressor protein from inhibiting transcription, thereby allowing transcription.

[0126] Any suitable transcriptional regulatory protein can be used, preferably a transcriptional regulatory protein that can be activated or inactivated. Preferably, an exogenous substance can be provided to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins.

[0127] Tetracycline-controlled transcriptional activation is a method of inducible gene expression in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcription activator protein is a tetracycline-responsive transcription activator protein (rtTa) or a derivative thereof. The rtTA protein is able to bind DNA at a specific TetO operator sequence. Several repeats of this TetO sequence are placed upstream of a minimal promoter (e.g., a CMV promoter), which together form a tetracycline response element (TRE). This system has two forms, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or inactivates (Tet-Off) the rtTA protein.

[0128] In the Tet-Off system, tetracycline or its derivatives bind to rtTA and inactivate rtTA, making it unable to bind to the TRE sequence, thereby preventing the transcription of the gene controlled by the TRE. This system was first described by Gossen et al. (1992).

[0129] The Tet-On system consists of two components; (1) a constitutively expressed tetracycline-responsive transcriptional activator protein (rtTa) and an rtTa-sensitive inducible promoter (Tet response element, TRE). This can be bound by tetracycline or its more stable derivatives including doxycycline (dox), resulting in activation of rtTa, causing it to bind to the TRE sequence and induce TRE-controlled gene expression. Use of this approach in the methods of the present invention may be preferred.

[0130] Therefore, the transcriptional regulatory protein can be a tetracycline-responsive transcriptional activator (rtTa) protein, which can be activated or inactivated by tetracycline or one of its derivatives, which is provided exogenously. If the transcriptional regulatory protein is rtTA, the inducible promoter inserted into the second GSH site includes a tetracycline response element (TRE). The exogenously provided substance is tetracycline or one of its derivatives. Variants and modified rtTa proteins can also be used in the methods of the present invention, including Tet-On advanced transactivators (also known as rtTA2S-M2) and Tet-On 3G (also known as rtTA-V16, derived from rtTA2S-S2).

[0131] The tetracycline response element (TRE) typically consists of seven 19 bp repeats of the bacterial TetO sequence separated by spacer sequences and with a minimal promoter. Variants and modifications of the TRE sequence are possible, as is the minimal promoter, which can be any suitable promoter. Preferably, the minimal promoter shows no or minimal expression levels in the absence of rtTa binding. The inducible promoter into which the second GSH is inserted can therefore comprise a TRE.

[0132] The modification system based on tetracycline control is the T-REX system, in which the transcriptional regulatory protein is the transcriptional repressor protein TetR. The components of this system include an inducible promoter and a Tet repressor (TetR), and the inducible promoter includes a strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites. In the absence of tetracycline, the Tet repressor forms a homodimer, which binds each TetO2 sequence in the inducible promoter with extremely high affinity and prevents the transcription of the promoter. Once added, tetracycline binds each Tet repressor homodimer with high affinity, so that it cannot bind the Tet operator. Then, the Tet repressor: tetracycline complex is separated from the Tet operator and allows induced expression. In this case, the transcriptional regulatory protein is TetR, and the inducible promoter includes two TetO2 sites. The exogenously provided substance is tetracycline or its derivatives.

[0133] The cumate switch is another approach to inducible gene expression, where transcription is reversibly turned on or off in the presence of cumate. The system can be used in both activator and repressor configurations, where the presence of cumate results in either repression of transcription or activation of transcription, respectively. In the repressor configuration, regulation is mediated by binding of the repressor (CymR) to an operator site (CuO) located downstream of a constitutive promoter. Addition of cumate, a small molecule, relieves repression and allows transcription to proceed. In the activator configuration, a chimeric transactivator (cTA) protein, formed by fusion of CymR to the VP16 activation domain, is able to activate transcription when bound to the CuO operator site, which is located upstream of the constitutive promoter. Addition of cumate abolishes DNA binding, thereby terminating transcription via cTA transactivation.

[0134] Thus, in some embodiments, the transcriptional regulatory protein may be a Tet-responsive transcriptional activator protein (rtTa) and / or a cumate repressor (CymR). In one embodiment, the expression of the lineage-specific transcription factor is controlled by a Tet response element. Thus, in another embodiment, the sequence encoding the lineage-specific transcription factor comprises a Tet-responsive element (TRE). In yet another embodiment, the expression of the sequence encoding the lineage-specific transcription factor is controlled (e.g., induced) by exogenous addition of tetracycline or its derivatives (e.g., doxycycline). In another embodiment, the expression of the one or more aging-inducing factors is controlled by a cumate switch. Thus, in yet another embodiment, the sequence encoding one or more aging-inducing factors comprises a CuO site. In another embodiment, the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is controlled by an exogenously provided substance such as tetracycline and its derivatives or cumate. In a specific embodiment, the expression of the aging-inducing factor is controlled, for example, by exogenous addition of cumate to induce.

[0135] Therefore, in other embodiments in which cells are somatic cells derived from iPSC by forward programming, the forward programming of iPSC to somatic cells is performed by expressing lineage-specific transcription factors after exogenous addition of tetracycline or its derivatives (e.g., doxycycline), and aging is induced by expressing aging-inducing factors after exogenous addition of cumate. In one embodiment, iPSC is forward programmed to somatic cells by expressing lineage-specific transcription factors after exogenous addition of tetracycline or its derivatives, and then aging is induced by expressing aging-inducing factors after exogenous addition of cumate. In an alternative embodiment, aging is induced by expressing aging-inducing factors after exogenous addition of cumate, and then iPSC is forward programmed to somatic cells by expressing lineage-specific transcription factors after exogenous addition of tetracycline or its derivatives.

[0136] Other inducible expression systems are known and can be used in the method of the present invention. These systems include a complete controlled inducible system from Agilent Technologies. This is based on the insect hormone ecdysone or its analog ponasterone A (ponA), which can activate transcription in mammalian cells transfected with the Drosophila melanogaster ecdysone receptor (EcR) gene and an inducible promoter comprising an ecdysone receptor binding site. EcR is a member of the retinoid-X-receptor (RXR) nuclear receptor family. In humans, EcR forms a heterodimer with RXR, which binds to an ecdysone response element (EcRE). In the absence of PonA, transcription is repressed by the heterodimer.

[0137] The transcriptional regulatory protein can be a repressor protein, such as CymR, ecdysone receptor or its derivatives. The latter example includes the VgEcR synthetic receptor of Agilent Technologies, which is a fusion of the DNA binding domain of EcR, glucocorticoid receptor and the transcriptional activation domain of herpes simplex virus VP16. The inducible promoter comprises an EcRE sequence or a modified form thereof and a minimal promoter. The modified form includes the E / GRE recognition sequence of Agilent Technologies, in which the sequence has been mutated. The E / GRE recognition sequence comprises the inverted half-site recognition elements of the retinoid-X-receptor (RXR) and GR binding domain. In all arrangements, the exogenously provided substance is ponasterone A, which removes the inhibitory effect of EcR or its derivatives on the inducible promoter and allows transcription to occur.

[0138] In one embodiment, the stem cell or somatic cell is from a mammal. In another embodiment, the mammal is a human. Therefore, in a specific embodiment, the stem cell or somatic cell is from a human and is a human stem cell or a human somatic cell, such as a somatic cell derived from a reprogrammed human iPSC. In an alternative embodiment, the mammal is a mouse, and the stem cell or somatic cell is optionally made to be a mouse stem cell or a mouse somatic cell, such as a somatic cell derived from a reprogrammed mouse iPSC.

[0139] In other aspects of the invention, artificial aged cells produced by the methods described herein are provided. In one aspect, artificial aged stem cells produced by the methods described herein are provided. Thus, in one embodiment, artificial aged stem cells are provided. In another aspect, artificial aged somatic cells derived from iPSCs by forward programming are provided, which are produced by the methods described herein. Thus, in another embodiment, artificial aged somatic cells are provided.

[0140] Application and screening methods

[0141] In another aspect of the present invention, there is provided the use of artificial aging cells as described herein in a method for identifying genes or gene combinations involved in reversing an aging phenotype or maintaining an unaged phenotype in a cell. In one aspect, there is provided the use of artificial aging stem cells as described herein in a method for identifying genes or gene combinations involved in reversing an aging phenotype or maintaining an unaged phenotype in a cell. In another aspect, there is provided the use of artificial aging somatic cells derived from iPSCs by forward programming as described herein in a method for identifying genes or gene combinations involved in reversing an aging phenotype or maintaining an unaged phenotype in a cell. In one embodiment, the use includes the use of artificial aging stem cells as described herein and artificial aging somatic cells derived from iPSCs by forward programming.

[0142] This use is based on the observation described herein that, after removing or reducing one or more aging-inducing factors, stem cells reverse the changes in the aging phenotype seen when aging is induced. Therefore, the unique regenerative ability of stem cells to actively reverse the induced aging phenotype can be used to screen for genes and gene combinations that participate in the reversal of the aging phenotype or the maintenance of the non-aging phenotype. In other words, by selectively targeting and inactivating, knocking out or activating genes suspected of participating in the reversal of the aging phenotype or the maintenance of the non-aging phenotype in stem cells, the effect of the targeting on the aging-induced method of the present invention can be observed.

[0143] Identification of genes and gene combinations can be carried out by sequencing. For example, targeted genes or gene combinations involved in the reversal of aging phenotypes or the maintenance of unaged phenotypes can be identified by transcriptome sequencing to determine the expression after targeting, such as expression levels. Alternatively, the gene or gene combination can be identified by detecting an agent for targeting the gene. In one embodiment, the gene or gene combination involved in the reversal of aging phenotypes or the maintenance of unaged phenotypes is identified by detecting a guide RNA for targeting the gene or detecting a plurality of guide RNAs for targeting the gene combination. In another embodiment, the guide RNA is detected by sequencing. In yet another embodiment, the guide RNA comprises a barcode sequence, and the detection of the guide RNA is carried out by sequencing using the barcode sequence.

[0144] Therefore, according to one aspect of the present invention, there is provided a method for identifying a gene or gene combination involved in reversing an aging phenotype or maintaining a non-aging phenotype in a cell, the method comprising the following steps:

[0145] i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time;

[0146] ii) optionally performing loss-of-function, inhibition, knockout, gain-of-function or a combination screening in said cell;

[0147] iii) measuring the senescence phenotype of said cells; and

[0148] iv) identifying genes or gene combinations as involved in reversal of an aged phenotype or maintenance of a non-aged phenotype when, upon loss-of-function, inhibition, knockout, gain-of-function or combination screening, the aged phenotype is altered and / or its progression is altered in cells compared to when the cells have not been screened.

[0149] In one embodiment, the method of identifying genes or gene combinations involved in reversing an aged phenotype or maintaining a non-aged phenotype in a cell using the artificial aged stem cells described herein and / or somatic cells derived from iPSCs by forward programming comprises the following steps:

[0150] i) performing loss-of-function, inhibition, knockout, gain-of-function or combined screening in the artificially aged stem cells or somatic cells;

[0151] ii) measuring the senescence phenotype of said cells; and

[0152] iii) identifying a gene or gene combination as being involved in the reversal of an aged phenotype or the maintenance of a non-aged phenotype when the aged phenotype in the cell is altered and / or its progression is altered when compared to an artificially aged stem cell or somatic cell that has not undergone loss-of-function, inhibition, knockout, gain-of-function or combined screening as defined herein.

[0153] In certain embodiments, the aging phenotype is as described herein. In one embodiment, the aging phenotype measured in the method is the transcriptome clock of the cell, optionally in combination with one or more other biological clocks (e.g., epigenetic clocks and / or ribosomal clocks) as described herein. In another embodiment, the gene or gene combination involved in the reversal of the aging phenotype or the maintenance of the non-aging phenotype is identified as described herein. In a specific embodiment, the gene or gene combination is identified by sequencing, as described herein.

[0154] In some embodiments, somatic cells are neurons, glial cells, blood cells, immune cells, liver cells, hepatocytes, muscle cells, myocytes, fibroblasts, skin cells, osteocytes, chondrocytes, epithelial cells, endothelial cells or adipocytes. In another embodiment, somatic cells are derived from iPSC by forward programming as described herein. In a specific embodiment, somatic cells are neurons derived from iPSC by forward programming. In another embodiment, wherein somatic cells are neurons as described herein, aging phenotypes can optionally be phenotypes of neurodegenerative diseases, such as reduced protrusion density and / or connectivity, shortened average dendrite length, shortened neurite diameter, neuronal marker gene downregulation or reduced electrophysiological activity.

[0155] In one embodiment, loss of function, inhibition, knockout, gain of function or combination screening includes nucleic acids, polypeptides (e.g., proteins, such as antibodies), aptamers and / or small molecules (e.g., small molecule compounds). Such nucleic acids, polypeptides, aptamers and small molecules include those that act as activators / agonists and those that act as inhibitors / antagonists. Therefore, it should be understood that the methods described herein can be used to screen for any regulatory factors (e.g., regulatory factors of expression or activity) of genes or gene combinations that are involved in reversing an aging phenotype or maintaining a non-aging phenotype in cells.

[0156] In another embodiment, loss of function, inhibition, knockout, gain of function or combination screening include whole-genome screening (whole-genome screen).This whole-genome screening, also referred to as whole-genome screening (genome-widescreen), is intended to be changed by changing the expression or activity of gene or gene product on a whole-genome scale and studying the obtained phenotypic changes, such as in the step iii) of the method described herein, to illustrate the relationship between genotype and phenotype.As the name implies, loss of function, inhibition or knockout screening disrupts the expression of gene, preventing them from functioning normally.Therefore, when loss of function, inhibition or knockout screening is performed, compared with the artificial aging cell in which loss of function, inhibition or knockout of gene or gene combination does not occur, when the aging phenotype continues for a longer time or is not reversed in the artificial aging cell after aging induction, then gene or gene combination is identified as participating in the reversal of aging phenotype. Alternatively, when loss of function, inhibition or knockout screening is performed, when the phenotype is lost in artificial aging cells, that is, compared with artificial aging cells in which loss of function, inhibition or knockout of genes or gene combinations does not occur, when aging cells show aging phenotypes after induction, then genes or gene combinations are identified as participating in the maintenance of unaged phenotypes. Functional gain screening enhances the activity or expression of genes or gene products. When using functional gain screening, compared with artificial aging cells in which the functional gain of the genes or gene combinations does not occur, when the aging phenotype in artificial aging cells that are aged after induction is reversed faster or lasts for a shorter period of time, then genes or gene combinations are identified as participating in the reversal of aging phenotypes. Alternatively, when the phenotype lasts in artificial aging cells, that is, compared with artificial aging cells in which the functional gain of genes or gene combinations does not occur, after induction of aging, the cells do not show aging phenotypes, then genes or gene combinations are identified as participating in the maintenance of unaged phenotypes. Combination screening includes one or more combinations of loss of function, inhibition, knockout and gain of function as described herein. Such combinatorial screening allows precise identification of combinations of genes involved in reversal of the aged phenotype or maintenance of the non-aged phenotype.

[0157] Methods for performing whole genome screening are known in the art, and any such methods will be understood to have practicality in the methods described herein. However, it is particularly useful to use CRISPR as described herein for screening. Therefore, in one embodiment, CRISPR is used for loss of function, inhibition, knockout, gain of function or combination screening. In another embodiment, wherein the screening is loss of function or inhibitory screening, the CRISPR is CRISPRi. CRISPRi can spatially inhibit transcription by blocking transcription initiation or extension. This is achieved by designing a single guide RNA (sgRNA) complementary to the promoter or exon sequence of the gene to be suppressed or repressed by transcription. According to the properties of the CRISPR effector, targeting the template strand or non-template strand leads to stronger repression. For example, for dCas9, when the guide RNA (gRNA) is complementary to the non-template strand, repression is stronger. Unlike transcription extension blocking, when targeting the transcription start site, the inhibition / repression of CRISPRi does not depend on the target DNA strand. In another embodiment, wherein the screening is knockout screening, the CRISPR is CRISPR-ko. CRISPR-ko involves the insertion or deletion of bases after the double-stranded DNA break repaired by the Cas9 nuclease, and the break is located in the gene targeted by the gRNA. After the double-stranded break is generated, an imprecise non-homologous end joining repair (NHEJ) approach is used, but it leads to the insertion or deletion of bases, thereby producing insertion / deletion or producing gene knockout. In another embodiment, wherein the screening is a functional gain screening, the CRISPR is CRISPRa. CRISPR activation (CRISPRa) uses a modified form of the CRISPR effector, which does not have endonuclease activity, but includes a transcriptional activator added to dCas9 and / or gRNA. Therefore, the transcriptional activator fused to the CRISPR component increases the expression of the gene after the target gene is targeted by gRNA. In another embodiment, wherein the screening is a combined screening, the CRISPR is a combination of two or more of the following: CRISPR, CRISPR-ko and CRISPRa, that is, a combination of two or more of any one of the above-mentioned CRISPR technologies.

[0158] Regeneration method

[0159] In another aspect of the present invention, a method for regenerating somatic cells or tissue-specific stem cells is provided, comprising changing the expression and / or activity of one or more genes identified by the methods described herein. It should be understood that "a kind" or "the" cell mentioned herein, such as a kind or the somatic cell, includes a single or a small amount of cells, and a cell group that may be larger in number. Therefore, it will be understood that, unless otherwise explicitly stated, any reference herein, including any aspect or embodiment, the singular includes the plural, and vice versa.

[0160] As used herein, the term "rejuvenating" refers to the reversal or maintenance of an aging phenotype, such as those described herein. Therefore, this regeneration will be understood as keeping the cell in a younger or non-aging state compared to a cell in a non-regenerated state, or will change the age of the cell so that it is younger or in a less aged state than before regeneration. Whether a cell is "younger," "non-aging," or "less aged" can be determined using any aging phenotype described herein, particularly using epigenetic and / or transcriptome clocks. For example, compared to cells in a non-regenerated state, regenerated somatic cells or tissue-specific stem cells may show less change in cell and / or nuclear morphology. Therefore, in one embodiment, the regenerated somatic cells or tissue-specific stem cells include a methylation age that is younger or younger than the somatic cells / tissue-specific stem cells in a non-regenerated state before regeneration (e.g., determined using an epigenetic clock). In another embodiment, the regenerated somatic cells or tissue-specific stem cells include a transcriptome age that is younger or younger than the somatic cells / tissue-specific stem cells in a non-regenerated state before regeneration (e.g., determined using a transcriptome clock). In another embodiment, the younger or less aged methylation age corresponds to the methylation age of somatic cells / tissue-specific stem cells at an earlier time point in the life cycle of a tissue or organism from which somatic cells are obtained. In another embodiment, the younger or less aged transcriptome age corresponds to the transcriptome age of somatic cells / tissue-specific stem cells at an earlier time point in the life cycle of a tissue or organism from which somatic cells are obtained. In another embodiment, with respect to the aging phenotype described herein, the regenerated somatic cells or tissue-specific stem cells include the phenotype of reprogrammed stem cells, such as pluripotent stem cells and / or iPSC. In some embodiments, the regenerated somatic cells or tissue-specific stem cells include the methylation age or transcriptome age corresponding to reprogrammed stem cells such as pluripotent stem cells and / or iPSC. Therefore, in one embodiment, the regenerated somatic cells or tissue-specific stem cells include the methylation age or transcriptome age of pluripotent stem cells. In another embodiment, the regenerated somatic cells or tissue-specific stem cells include the methylation age or transcriptome age of iPSC. In another embodiment, the regenerated somatic cells or tissue-specific stem cells include the methylation age and transcriptome age of pluripotent stem cells. In another embodiment, the regenerated somatic cell or tissue-specific stem cell comprises the methylation age and transcriptomic age of the iPSC.

[0161] Any method known in the art for regulating and / or changing the expression and / or activity of one or more genes or gene combinations identified by the methods described herein can be used.For example, when expression and / or activity are to be increased, the expression / activity induction using one or more expression cassettes described herein or by introducing nucleic acids, polypeptides or small molecules (such as activators / agonist polypeptides or small molecules) can be utilized.Alternately, when expression and / or activity will be reduced and / or repressed, any inhibitory small molecules, antagonistic polypeptides, interfering nucleic acids or interference / knockout gene editing methods can be used.These methods and approaches are known in the art and described herein.In a specific embodiment, CRISPR is used, for example, by CRISPRi and / or CRISPRa to change the expression of one or more genes or gene combinations identified by the methods described herein.It should be understood that in the case of a gene combination, the expression of a gene or a group of genes can be increased, while the expression of another gene or another group of genes is suppressed / repressed. In one embodiment, the expression of one or more genes or gene combinations involved in the reversal of an aging phenotype or the maintenance of an unaged phenotype (i.e., promoting / positively affecting the reversal or maintaining and promoting a young, unaged or regenerative state) is found to be increased, for example, activated or enhanced. In another embodiment, CRISPRa is used to increase expression. In another embodiment, the expression of one or more genes or gene combinations that promote an aging phenotype or accelerate the loss of an unaged phenotype (i.e., promote aging) is found to be reduced and / or inhibited. In yet another embodiment, CRISPRi and / or CRISPR-ko are used to reduce and / or inhibit expression. In some embodiments, the expression of a gene combination is increased (e.g., activated or enhanced) and reduced and / or inhibited.

[0162] In some embodiments, the regenerated somatic cells include a reduction and / or slowing of the progression of an aging phenotype compared to a non-regenerated somatic cell, wherein the aging phenotype is as described herein. In another embodiment, the regenerated somatic cells do not include an aging phenotype found in a somatic cell in a non-regenerated state. For example, a somatic cell may include an aging phenotype as described herein before regeneration, and the aging phenotype is eliminated after regeneration.

[0163] In some embodiments, the somatic cell is a neuron, a glial cell, a blood cell, an immune cell, a liver cell, a hepatocyte, a muscle cell, a myocyte, a fibroblast, a skin cell, a bone cell, a chondrocyte, an epithelial cell, an endothelial cell, or a fat cell. In another embodiment, the somatic cell is derived from iPSC by forward programming as described herein.

[0164] It is easy to understand that the methods and uses described herein or any / all steps thereof can be performed in vivo, ex vivo or in vitro. In a specific embodiment, the methods and uses described herein or any / all steps thereof are performed in vitro, such as ex vivo.

[0165] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. As used herein, the term "about" used herein includes up to 10% (including 10%) greater than the specified value and up to 10% (including 10%) less than the specified value, appropriately up to 5% (including 5%) greater than the specified value, especially up to 5% (including 5%) less than the specified value. As used herein, the term "between" includes the value of the specified boundary.

[0166] It should be understood that all embodiments described herein may be applicable to all aspects of the invention, and vice versa.

[0167] Other features and advantages of the present invention will be apparent from the description provided herein.It should be understood, however, that the description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only, as various changes and modifications will become apparent to those skilled in the art.

[0168] Terms

[0169] A set of clauses defining the present invention, its aspects and embodiments is as follows:

[0170] 1. A method for transiently inducing aging in stem cells, the method comprising:

[0171] (i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0172] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0173] 2. The method according to clause 1, wherein the stem cell is a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a germ stem cell, a multipotent stem cell, an oligopotent stem cell, a unipotent stem cell or a tissue-resident stem cell.

[0174] 3. A method for transiently inducing aging in somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising:

[0175] (i) before or after forward programming of iPSCs, introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and

[0176] (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

[0177] 4. The method according to clause 3, wherein the somatic cell is a neuron, a glial cell, a blood cell, an immune cell, a hepatocyte, a muscle cell, a myocyte, a fibroblast, a skin cell, a bone cell, a chondrocyte, an epithelial cell, an endothelial cell or an adipocyte.

[0178] 5. The method according to any one of clauses 1 to 4, wherein the introduction of the one or more aging-inducing factors in step (i) comprises one or more expression cassettes comprising a sequence encoding the one or more aging-inducing factors.

[0179] 6. The method according to clause 5, wherein one or more expression cassettes are transiently transfected into the cells.

[0180] 7. The method according to clause 5, wherein the expression cassette and the gene encoding the transcriptional regulatory protein are inserted into a genetic safe harbor site,

[0181] The expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein.

[0182] 8. The method according to clause 5, wherein the gene encoding the transcriptional regulatory protein is inserted into the first gene safe harbor site and the expression cassette is inserted into the second gene safe harbor site,

[0183] wherein the expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein, and

[0184] The first genetic safe harbor site and the second genetic safe harbor site are different.

[0185] 9. The method according to any one of items 5 to 8, wherein the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is controlled by an exogenously provided substance such as tetracycline and its derivatives or cumate.

[0186] 10. The method according to any one of clauses 1 to 7, wherein the one or more aging-inducing factors is an alternatively spliced ​​form of lamin A, such as presenilin.

[0187] 11. The method according to any one of clauses 1 to 10, wherein the one or more aging-inducing factors are mitochondrial DNA mutations, such as deletion of DNA polymerase subunit gamma (POLG).

[0188] 12. A method according to any one of clauses 1 to 11, wherein the one or more aging-inducing factors are aging-inducing compounds, such as telomerase inhibitors, reactive oxygen species (ROS) or compounds that disrupt or stress mitochondrial function, such as 6-hydroxydopamine (6-OHDA), valinomycin, CCCP, rotenone or hydrogen peroxide.

[0189] 13. The method according to any one of clauses 1 to 12, wherein the one or more aging-inducing factors are inducers of DNA damage, such as ionizing radiation, platinum-containing drugs, cyclophosphamide, chlorambucil or temozolomide.

[0190] 14. An artificial aged stem cell or a somatic cell derived from iPSC by forward programming, produced by the method of any one of clauses 1 to 13.

[0191] 15. Use of the artificial aged stem cells and / or somatic cells derived from iPSCs by forward programming according to Item 14 in a method for identifying genes or gene combinations involved in reversal of aged phenotype or maintenance of non-aged phenotype in cells.

[0192] 16. A method for identifying a gene or gene combination in a cell that is involved in reversing an aging phenotype or maintaining a non-aging phenotype, the method comprising the following steps:

[0193] i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time;

[0194] ii) optionally performing loss-of-function, inhibition, knockout, gain-of-function or a combination screening in said cell;

[0195] iii) measuring the senescence phenotype of said cells; and

[0196] iv) identifying genes or gene combinations as involved in reversal of an aged phenotype or maintenance of a non-aged phenotype when, upon loss-of-function, inhibition, knockout, gain-of-function or combination screening, the aged phenotype is altered and / or its progression is altered in cells compared to when the cells have not been screened.

[0197] 17. A method according to clause 16, wherein the loss-of-function, inhibition, knockout, gain-of-function or combined screening comprises a genome-wide screening.

[0198] 18. A method according to clause 16 or clause 17, wherein CRISPR is used for loss-of-function, inhibition, knockout, gain-of-function or combined screening.

[0199] 19. The method of clause 18, wherein the screen is a loss-of-function or inhibition screen and the CRISPR is CRISPRi, a knockout screen and the CRISPR is CRISPR-ko, a gain-of-function screen and the CRISPR is CRISPRa, or a combination screen and the CRISPR is a combination of two or more of: CRISPRi, CRISPR-ko, and CRISPRa.

[0200] 20. The use according to clause 15 or the method according to any one of clauses 16 to 19, wherein the aging phenotype of the cell is selected from one or more of the following: proliferation, senescence, changes in cell and / or nuclear morphology, mitochondrial function, changes in gene expression, upregulation of lamin A and / or lamin C nuclear proteins, epigenetic marks associated with aging, changes in DNA or histone methylation, methylation entropy, DNA double-strand breaks, telomere length, and transcriptome and / or epigenetic clocks.

[0201] 21. The use or method according to clause 20, wherein the aging phenotype is decreased proliferation and / or increased senescence,

[0202] wherein the changes in nuclear morphology are abnormal folding, blebbing and / or loss of nuclear circularity,

[0203] wherein the mitochondrial function is reduced oxygen consumption and / or increased mitochondrial reactive oxygen species (ROS),

[0204] wherein the changes in gene expression are selected from one or more of the following: downregulation of somatic lineage identity genes, downregulation of mitochondrial genes, upregulation of apoptosis and / or aging-related genes, and upregulation of DNA damage-related genes,

[0205] wherein the epigenetic markers associated with aging are selected from one or more of the following: reduced heterochromatin trimethylation H3K9 (H3K9me3), reduced HP1γ and increased γH2AX,

[0206] where telomere length is shortened, and / or

[0207] Wherein the transcriptome and / or epigenetic clock is a single cell transcriptome and / or epigenetic clock.

[0208] 22. The use or method according to any one of clauses 15 to 21, wherein the cell is a neuron derived from iPSC by forward programming, and optionally wherein the aging phenotype is a phenotype of a neurodegenerative disease, such as reduced neurite density and / or connectivity, reduced average dendrite length, reduced neurite diameter, downregulation of neuronal marker genes, or reduced electrophysiological activity.

[0209] 23. A method of regenerating a somatic cell comprising altering the expression and / or activity of one or more genes identified by the use or method according to any one of clauses 15 to 22.

[0210] 24. The method according to clause 23, wherein the regenerated somatic cell comprises a reduced and / or slower progression of an aging phenotype compared to a non-regenerated somatic cell, and wherein the aging phenotype is as defined in clause 20 or clause 21.

[0211] 25. A method or use according to any one of clauses 1 to 13 or clauses 15 to 24, wherein the method or use is performed in vitro.

[0212] The invention will now be described using the following non-limiting examples:

[0213] Example

[0214] Example 1 - Materials and Methods

[0215] hiPSC culture

[0216] Human induced pluripotent stem cells Bob (A1ATD-iPSC) (Yusa et al., 2011) and KOLF2 (HPSI0114i; Human Induced Pluripotent Stem Cell Project, www.hipsci.org) were cultured as previously described (Pawlowski et al. (2017); and Vallier 2011). Briefly, cells were seeded on Geltrex-coated culture dishes and cultured in essential E8 medium (Life Technologies). Cells were passaged in small clumps every 6-7 days using ReLeSR (StemCellTechnologies). To age NGN2-Progerin iPSCs, cumate (System Biosciences) was added to the culture medium for 5 days at 60% confluence, after which cumate was withdrawn and iPSCs were isolated for induction.

[0217] Gene targeting constructs and molecular cloning

[0218] For the initial constructs based on the OPTi-OX system, the previously designed vector pR26_CAG-rtTA was used (Pawlowski et al. (2017) and WO 2018 / 096343, the sequences of which are incorporated herein by reference). For the inducible AAVS1 targeting vector, GFP-Progerin (IDT) was cloned into the previously designed pAAV_TRE-NGN2 vector (WO 2018 / 096343, the sequences disclosed therein are incorporated herein by reference) via the P2A site, or into pAAV_HS4 (provided by Professor Cédric Ghevaert). To construct the dual inducible system (OPTi-OX) 2 , the pR26_CAG-rtTA-CymR targeting vector was constructed by ligating the cumate repressor CymR (Addgene, plasmid 48099) into our original pR26_CAGrtTA vector via the P2A site. For the inducible AAVS1 targeting vector, the aging vector was constructed using the original pAAV_TRE-NGN2 vector. After separating the NGN2 fragments by the STOP codon and the polyA sequence, GFP-presenilin and the cumate operon CuO (Addgene, plasmid 48099) were ligated by Gibson Assembly (New England Biolabs).

[0219] HEK293T culture and transfection

[0220] Human embryonic kidney 293T (HEK 293T) cells were cultured in DMEM-high glucose supplemented with 5% fetal bovine serum (FBS). pR26_CAG-rtTA-CymR and pAAVS1_NGN2-presenilin targeting vectors were transiently co-transfected into 293T cells in the presence of a lipid transfection reagent (ThermoFisher Scientific). 2 million 293T cells were plated on each 6-well plate for transfection. The next day, when the cells reached 50-70% confluence, a transfection mixture consisting of OPTI-MEM medium (Gibco), a lipid transfection reagent, a targeting vector, two gRNAs, and two ZFNs was prepared and added to the plated 293T cells. The next day, the cells were carefully washed once with PBS, and then the culture medium was replaced with DMEN-high glucose containing 5% FBS in the presence or absence of doxycycline and / or cumate. The next day, the cells were observed under an EVOS FL microscope (Life Technologies).

[0221] Gene targeting

[0222] Targeting of the hROSA26 and AAVS1 loci was performed as previously described (Bertero et al. (2016), Pawlowski et al. (2017), and WO 2018 / 096343, the sequences and methods of which are incorporated herein by reference). Targeting of the hROSA26 locus was performed by nuclear transfection. Neomycin-resistant colonies were selected and screened by genotyping. The correct clones targeting hROSA26-rtTA-CymR were then targeted by nuclear transfection with an inducible transgene cassette in the AAVS1 locus. The resulting puromycin-resistant colonies were selected and reanalyzed by genotyping.

[0223] Targeting of the hROSA26 locus (Irion et al. (2007)) was performed by nuclear transfection. Human induced PSCs were isolated into single cells using Accutase (Gibco) and 2 × 10 cells were transfected with 4D-NucleofectorX kit using Lonza P3 Primary Cell 4D-NucleofectorX kit and circulating CA-137 of Lonza 4D-Nucleofector System. 6 Cells were nucleofected (100 μL reaction volume; a total of 12 μg DNA, which was evenly divided between the two gRNA / Cas9n plasmids and the targeting vector). The nucleofected hiPSCs were seeded into Geltrex (Thermo Fisher Scientific)-coated culture dishes and cultured in essential E8 medium. Colne-R (StemCell Technologies) was added 48 hours after nucleofection to promote cell survival. After 3-4 days, neomycin-resistant hiPSCs were selected by adding G418 (50 μg / mL, Sigma-Aldrich) for 7 days. Subsequently, individual clones were picked, expanded, and finally analyzed by genotyping.

[0224] Targeting of the AAVS1 locus (Hockemeyer et al., 2009) was performed by nuclear transfection similar to the hROSA26 locus (100 μL reaction volume; a total of 12 μg DNA, which was evenly divided between the two AAVS1 ZFN plasmids and the targeting vector). After 3-4 days, resistant hiPSCs were selected by adding puromycin (1 μg / mL, Sigma-Aldrich) for 4-5 days. Subsequently, individual clones were picked, expanded, and analyzed by genotyping.

[0225] Drug-resistant hiPSC clones from targeted experiments were screened by genomic PCR to verify site-specific transgene integration, to determine the number of targeted alleles, and to exclude off-target integration. PCR was performed with LongAmp Taq DNA polymerase (New England Biolabs).

[0226] Generation of iNeurons from hiPSCs and aging of iNeurons

[0227] NGN2 and NGN2-progerin iPSCs were dissociated into single cells using Accutase and plated at 30K cells / cm 2 The cells were seeded at a density of 100 × 20 μg / mL on PDL / Geltrex coated culture dishes. Forward programming was initiated 24 hours after the split. Induction was performed in DMEM / F-12 supplemented with Glutamax (100×), non-essential amino acids (100×), 50 μM 2-mercaptoethanol (Gibco), 1% penicillin / streptomycin, and 1 μg / mL Dox. After 2 days of induction, the culture medium was changed to Neurobasal-medium supplemented with Glutamax (100×), B27 (50×), 10 ng / mL BDNF (Peprotech), 10 ng / mL NT3 (R&D Systems), 1% penicillin / streptomycin and 1 μg / mL Dox. In order to age the NGN2-presenilin iNeuron (iN), cumate was added to the culture medium for 7 days at D7 after induction. Pre-aging iNs were induced from 5-day-aged NGN-presenilin iPSCs as described above, and no additional cumate was added during the induction process.

[0228] Primary rodent astrocyte cultures for Mito stress testing

[0229] All experiments on animals or involving the use of animal tissue were performed under a project license under the Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 and were ethically reviewed by the University of Cambridge Animal Welfare and Ethical Review Committee.

[0230] Primary mixed glial cultures were derived from the cerebral cortex of P0-P2 newborn Spraque Dawley rats and generated according to previous guidelines (McCarthy and De Vellis (1980)) and slightly modified (Syed et al. (2008)). The mixed glial cells were maintained in culture for 10 days, after which the flasks were shaken at 260 rpm for 1 hour on an orbital shaker to remove loosely attached microglia, and then at 260 rpm overnight to remove oligodendrocyte precursors. Astrocyte cultures were then maintained in glial culture medium (Dublin's modified eagle medium (DMEM), supplemented with 10% FBS, glutamine and 1% penicillin / streptomycin) for at least two weeks before passage and seeding with iN for multi-electrode array recording.

[0231] Immunocytochemistry

[0232] Cells were fixed in 4% paraformaldehyde (Alfa Aesar) at room temperature for 15 minutes and then washed three times with PBS. The cells were then permeabilized with 0.1% Triton-X-100 for 15 minutes at room temperature. Then, 10% goat serum or donkey serum (abcam) and 0.3% Triton X-100 (diluted in PBS) were blocked at room temperature for 30-45 minutes. Subsequently, the cells were incubated overnight at 4°C with the first antibody appropriately diluted in 2% goat serum or donkey serum and 0.1% Triton X-100 (diluted in PBS). After washing three times with PBS, the cells were incubated with the corresponding secondary antibodies in PBS supplemented with 1% goat serum or donkey serum for 1 hour at room temperature. The nuclei were observed by 4',6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific).

[0233] Images were acquired using a Zeiss LSM 700 confocal microscope (Leica). For quantification of cellular fluorescence intensity, images were processed using ImageJ software (version 2.00, NIH). Corrected whole-cell fluorescence (CTCF) was assessed for each tested marker of the cells and presented as a bar graph. The number of γH2Ax foci was manually counted and plotted as a bar graph.

[0234] Neurite measurement

[0235] Dendritic length was measured using ImageJ software with the NeuronJ plugin to trace MAP2-labeled neurites and is presented as a bar graph. Neurite diameter was measured using ImageJ software and is presented as a scatter plot and a Gaussian distribution plot using nonlinear regression.

[0236] Cellular Mito stress test

[0237] iN, post-aging iN and pre-aging iN were induced in culture for 3 days, then separated into single cells using Accutase and re-seeded on Seahorse XFp cell culture mini microplates (Agilent). Induction was performed as described above. On the 14th day after induction, the induction medium was replaced with XF DMEM medium containing 10mM glucose, 1mM pyruvate and 2mM L-glutamine before the experiment. Mitochondrial function was assessed using the Cell Mito Stress test kit (Agilent). Cells were treated with 10μM oligomycin, 10μM carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) and 5μM rotenone / antimycin A. Oxygen consumption rate (OCR) was measured at 37°C using the Mito Stress Test program using a Seahorse XFp extracellular flux analyzer (Agilent).

[0238] Multi-electrode array (MEA) recordings

[0239] Non-senescent (NGN2) iNs, post-senescent and pre-senescent (N2P) iNs were dissociated into single cells at day 3 post-induction and replated into PDL / Geltrex-coated wells of CytoView MEA 48 (Axion Biosystems) at a 1:1 ratio with or without rat cortical astrocytes. Co-cultures were maintained in Neurobasal medium during recording. Recordings were performed at D14 and D21 after induction of iNeurons using the Maestro Pro MEA system (Axion Biosystems).

[0240] RNA extraction

[0241] Total RNA was extracted from hiPSCs and iNeurons in each condition using the GenElute mammalian total RNA miniprep kit (Sigma-Aldrich) and On-Column DNaseI Digestion Set (Sigma-Aldrich).

[0242] Real-time quantitative PCR (RT-PCR)

[0243] cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) was used for RT-PCR. Samples were run on a QuantStudio 6Flex Real-Timer PCR System machine (Applied Biosystems). All samples were analyzed in technical replicates and normalized to the housekeeping gene porphobilinogen deaminase 1 (PBGD1). Results were analyzed using the ΔΔCt method.

[0244] RNA sequencing

[0245] Read preprocessing, QC, alignment, and gene quantification. Before differential expression (DE) analysis, raw reads were evaluated for quality checks in terms of sequencing quality and contamination. Trimming was performed using BBDuk [from BBMap v38.76] (Bushnell, 2017). Reads were then aligned to the latest reference genome (hg38) with GTF from Gencode (v33) using STAR aligner v2.7.1a (Dobin et al., 2013). Aligned reads were converted to read counts per gene using the RSEM v1.3.3 tool (Li & Dewey, 2011).

[0246] Differential expression analysis. Paired expression analysis was performed on multiple sample combinations based on control, aging, and iN / iPSC using the DESeq2 R package (Love et al., 2014). In short, expression counts were scaled and normalized to correct for sequencing depth of paired groups and batch differences between samples. These standardized counts were then used for differential expression analysis, and fold change values ​​for comparison were generated in log2 ratio [log2 (sample 2 / sample 1)]. Genes with lower read counts can produce higher fold change values, which can lead to possible false positives. Therefore, in order to regulate the fold change that is increased due to the ratio between lower read counts in the sample, the fold change shrinkage estimation method from DESeq2 was used. Genes with adjusted p-values ​​≤ 0.05 and log2 fold changes of +1 or -1 were considered as significantly upregulated and downregulated genes. In addition, genes were scored based on P value, average TPM, and logFC using the desiR v1.2.1R package (Lazic, 2015) to prioritize highly significant genes.

[0247] Annotations and interpretations. Principal component analysis (PCA) and volcano plots were generated for different groups and conditions using DESeq2 and ggplot2 (Wickham, 2016). Venn diagrams were generated by calculating the intersection / overlap of significant DE genes in different comparisons using the VennDiagram v1.6.20 R package (H. Chen & Boutros, 2011). Heatmaps of marker genes and gene ontology (GO) specific genes were drawn using the ComplexHeatmap v2.2.0 R package (Gu et al., 2016). GO analysis was performed on up-regulated and down-regulated genes from different analyses using the EnrichR v2.1 R package (EY Chen et al., 2013). Statistically enriched biological processes were considered (adjusted P value <= 0.05).

[0248] DNA methylation

[0249] Genomic DNA was extracted from the different samples using the Qiagen DNeasy blood and tissue kit according to the manufacturer's protocol. The samples were sent to the Illumina InfiniumMethylationEPIC BeadChip array for DNA methylation services via Erasmus MC (University Medical Center Rotterdam, the Netherlands).

[0250] Epigenetic clock analysis

[0251] Steve Horvath's team performed epigenetic clock analysis on the raw DNA methylation data based on their previously published research results (Horvath 2013).

[0252] Transcriptome clock analysis

[0253] Transcriptome clock analysis was performed on RNA raw data using ShiftBioscience's proprietary algorithm.

[0254] Entropy analysis

[0255] Shannon entropy analysis was performed as previously described (Hannum et al. (2013), Molecular Cell). The following formula was used:

[0256]

[0257] where MFi is the methylation fraction of the i-th methylated marker and N is the number of markers.

[0258] Statistical analysis

[0259] Statistical analysis was performed using GraphPad Prism (v8). The number of replicates, statistical tests used, and test results are described in the figure legends. Unless otherwise stated, data are expressed as mean ± SEM. Two-group comparisons were analyzed using Student's t test. For comparisons between two groups, unpaired student's t test was used. For multiple comparisons, one-way analysis of variance (ANOVA) and Dunnett's or Tukey's multiple comparison tests were used.

[0260] Engineering of Cas9 lines for CRISPR screening

[0261] Three transgenes were integrated into GSH by genome engineering. First, a CAG promoter-driven rtTA cassette was integrated into the ROSA26 locus for transcriptional activation of the second recombinant transgene controlled by doxycycline. Second, a fusion protein of GFP and presenilin under the control of a doxycycline-responsive promoter was integrated into the AAVS1 locus. Finally, a CAG promoter-driven full-length Cas9 protein was integrated into the CLYBL locus, upstream of the P2A ribosomal skipping sequence and the fluorescent protein mCherry, for verification of integration.

[0262] CRISPR Screening

[0263] A CROP-seq screen (Datlinger et al. (2017) Nature Methods) was performed to ensure genotype-phenotype associations in downstream analyses, i.e., changes in the transcriptome of a single cell could be traced back to the presence of certain sgRNAs and, therefore, to the KO of a gene. An engineered hiPSC line (KOLF2-GFP-Progerin-Cas9) was transduced with lentivirus encoding a library of sgRNAs targeting 1,000 genes at a low MOI (MOI < 0.1) to ensure single integration of a single sgRNA. Subsequently, transduced cells were selected by puromycin treatment, which enabled the puromycin resistance cassette present in the CROP-seq vector. Engineered hiPSC lines containing single gene knockouts were treated with doxycycline for 5 days to induce a progerin-mediated aging phenotype, followed by a 14-day regeneration period (i.e., removal of doxycycline). At the end of the time course, regenerated iPSCs were processed as single cells for subsequent scRNA-seq analysis using 10× Genomics Chromium 5'Chemistry.

[0264] Identification of gene knockouts affecting aging / regeneration

[0265] In order to identify the impact of single gene knockout on the aging / regeneration of hiPSC at the single cell level, a linear discriminant analysis (LDA) using aging features was performed to establish an aging scale. In other words, aging features are used to generate a continuous variable (i.e., aging score) describing the "aging" degree of a single cell at the transcriptional level. The aging scale is integrated into the unified manifold approximation and projection (UMAP) diagram of all single cells processed in CRISPR knockout screening. The scale is used to evaluate the impact of knockout on the regeneration process compared to cells transduced with non-targeted sgRNA ("non-targeted") used as a control.

[0266] Example 2 - Aging and regeneration of aged hiPSCs

[0267] iPSC N2P The system can study three aging modes ( Figure 1 A): 1) "Pre-aging" of cells at the iPSC stage, which can subsequently be converted into neurons; 2) "Post-aging" of cells at the iNeuron stage, and finally; 3) aging of iPSCs without conversion into adult iNeurons. A fundamental difference in this model is that the cells are in a post-mitotic stage during the post-aging stage. It was therefore hypothesized that iPSCs and iNs might respond differently to progerin expression, which could account for the differences between "pre" aged iNs and "post" aged iNs. Since iPSCs reset their aged identity during the reprogramming process, it was tested whether this system could induce aging-associated features in these cells, using iPSCs that do not overexpress progerin NGN2 In addition, iPSCs expressing progerin only under the TetOn system were generated, termed Bob 早老蛋白 and Kolf 早老蛋白 ( Figure 1 B) Progerin over 5 days induces typical morphological changes observed in the nuclei of HGPS patients. N2P / 早老蛋白 Cells or control iPSCs NGN2 Compared to aged iPSCs N2P and iPSCs 早老蛋白 Cells showed abnormal nuclear morphology, such as folding, blebbing, and loss of circularity ( Figure 1 CI and Figure 2 AB). In addition, we investigated epigenetic marks associated with physiological aging. We found that aged iPSCs showed a significant loss of heterochromatin trimethylated H3K9 (H3K9me3), which is usually reduced with physiological aging, and increased DNA double-strand breaks ( Figure 3 and 4Another phenotype associated with aging is senescence. Senescence was tested by staining for the senescence-activated β-galactosidase (SA-β-gal) marker in aged iPSCs, and the results showed that SA-β-gal was significantly increased in aged iPSCs ( Figure 5 ) To test whether the cells maintain their aging phenotype, aged iPSCs were passaged. After the first passage after aging, iPSCs appeared to regenerate and the aging phenotype was reversed, as evidenced by their normal nuclear morphology, H3K9me3 expression levels, and number of DNA double-strand breaks ( Figure 1 CG, 2, 3 and 4).

[0268] Example 3 - Development of a dual inducible system (OPTI-Ox) 2 , for artificially aged iPSCs and iNeurons

[0269] As a first principle proof, a test mode using a single inducible gene switch based on a single box combining the expression of progerin and NGN2 was investigated. To this end, a previously published protocol for inducing neurons based on the OPTi-OX system was used. This system has been shown to positively program hiPSCs to functional cortical neurons by overexpressing NGN2 from a genetic safe harbor (Pawlowski et al. (2017)). These induced neurons (iNeurons) represent a homogenous population of glutamatergic neurons and have been extensively characterized, showing expression of relevant neuronal markers and having electrophysiological properties (Lam et al. (2017); Pawlowski et al. (2017); Tourigny et al. (2018); and Zhang et al. (2013)). An iNeuron (iN) line expressing GFP fused to progerin was generated to allow the expression of progerin in the cells. In this box, the reprogramming factors and progerin are connected by a 2A sequence ( Figure 6 A). Once the reprogramming factor NGN2 is expressed, the cassette ensures efficient transgenic expression of progerin. It was found that progerin was expressed efficiently in the generated neurons. Compared with control NGN2-iNs, these iNs displayed artificial aging phenotypes such as changes in nuclear morphology and upregulation of lamin A and lamin C ( Figure 6 BC). In addition, mitochondrial dysfunction was observed, as reflected by a decrease in oxygen consumption levels ( Figure 6 D). However, cell viability is impaired at an early stage ( Figure 6E). Titration of doxycycline concentrations used to induce NGN2-presenilin expression suggests that this may be a result of the high levels of presenilin produced by this method (data not shown). Therefore, it would be more informative to hypothesize that a new system is needed to express NGN2 and presenilin at different time points.

[0270] To allow for separate control of NGN2 and progerin expression, we used OPTi-OX technology and designed a dual system based on the inducible systems of TetOn (Baron and Bujard (2000)) and cumate switch (Mullick et al. (2006); Sato et al. (2020); and Seo and Schmidt-Dannert (2019)). This new system is called (Opti-OX) 2 The cumate repressor (CymR) was first cloned into the original ROSA26 targeting vector and linked to rtTA via the 2A sequence ( Figure 7 AF). Next, the cumate operon (CuO) and subsequently GFP-presenilin were cloned into the original targeting cassette for cortical neurons (AAVS1-NGN2; Figure 7 AF). Initially, two constructs were tested for the AAVS1 locus. The first construct oriented the expression cassette in opposite orientations to mitigate potential leakiness of the NGN2 promoter (referred to as "reverse"), whereas the second construct oriented the presenilin and NGN2 expression cassettes in the same orientation (referred to as "forward"; Figure 7 D). Both AAVS1 constructs were transfected into HEK293 cells along with the rtTA-CymR construct. The cells were then treated with cumate, demonstrating successful induction of progerin in AAVS1-NGN2-progerin "forward" transfected cells, but not in "reverse" transfected cells (data not shown). Subsequently, NGN-progerin (N2P) lines were generated in the Bob and KOLF2 iPSC backgrounds using rtTA-CymR and AAVS1-NGN2-progerin "forward" constructs. Drug-resistant hiPSC clones from both lines were screened by genomic PCR to verify site-specific transgene integration, to determine the number of targeted alleles, and to exclude off-target integration ( Figure 7 E) To evaluate the iPSCs obtained N2P To determine whether the cells were able to generate neurons, doxycycline was added to the culture medium without activating the cumate system. Both lines showed neuronal development and neuronal network formation similar to the original NGN2-iN system ( Figure 7F). In addition, no differences were observed in NGN2 overexpression based on RT-PCR analysis (data not shown). In a series of extensive experiments, the dosing regimen and cumate concentration were varied to optimize the expression level of progerin ( Figure 7 GJ). GFP expression was detected in neurons induced with both doxycycline and cumate, but not in neurons induced with doxycycline alone ( Figure 7 H). In addition, GFP expression can be titrated by adjusting the dose of cumate ( Figure 7 I) Due to cellular autofluorescence, slight background expression can be detected from cells that do not contain cumate, e.g. Figure 7 As shown by HI.

[0271] Example 4 - Pre-aging and post-aging iNeurons exhibit aging phenotypes

[0272] Next, we tested two other models, pre-aging iN (aiN) and post-aging iN (iaN). To this end, Bob and KOLF iPSCs were N2P Target cells and iPSCs NGN2 Cells were treated with doxycycline for 7 days in chemically defined neuronal medium. N2P Cells were aged prior to inducing NGN2 expression as described above. To induce senescence in the post-senescence group, cumate was added to the culture medium on day 7 after induction for one week. Following progerin overexpression, aged iNs (pre-senescence and post-senescence) showed enhanced signs of nuclear folding and blebbing at both day 14 and day 21 after induction based on GFP and lamin A expression, whereas control iN nuclei remained intact ( Figure 8 AB). Interestingly, GFP expression was also observed for presenescent iNs at days 14 and 21 after induction, even though they were only treated with cumate at their iPSC stage, suggesting a slow turnover of prosenescent proteins in the nuclear membrane. Immunostaining for the lamina-associated protein LAP2a also showed a significant decrease in expression in presenescent and postsenescent iNs compared to control iNs ( Figure 8 CE and 8G). Similarly, like aged iPSCs, it was also found that the nuclear morphology of aged iNs was less round and more elongated ( Figure 8 F and 8H). Then, parameters related to degenerative changes in neural network complexity were examined. It was found that aged iNs were significantly different from control non-aged iNs ( Fig. 9 A) shows reduced neurite density and connectivity compared to iNs. The reduction in neurite complexity is reflected in a significant decrease in mean dendritic length (based on MAP2 expression) and a decrease in neurite diameter in aged iNs ( Fig. 9This was supported by quantitative RT-PCR analysis, which showed downregulation of several neuronal markers (i.e., MAP2, vGLUT2, synapsin-1, synaptophysin, and PSD95) in aged iNs, and ICC, which showed downregulation of the synaptic marker synapsin-1 ( Fig. 9 FG). To eliminate the possibility that cumate itself caused these changes, iPSCs treated with and without cumate were NGN2 - and iPSCs N2P -derived iN were subjected to RT-PCR analysis. These data confirm that the reduction in neuronal markers is due to progerin overexpression and not to exposure to cumate molecules ( Fig.10 ).

[0273] Example 5 - Peripheral heterochromatin mark expression and DNA damage in aged iNeurons

[0274] To further characterize the aging-like phenotype of iNs after progerin overexpression, the expression of two heterochromatin markers, H3K9me3 and HP1γ, was evaluated. Notably, both markers were significantly downregulated in aged neurons at days 14 and 21 after induction ( Fig.11 AF). These aging-like phenotypes have not been observed previously in neurons overexpressing progerin (Miller et al. (2013)). In addition, based on γH2AX staining, the formation of DNA double-strand breaks, an important hallmark of physiological aging, was observed in aged neurons, with aged iNs showing a higher percentage of cells with >5 lesions than control non-aged iNs ( Fig.11 G).

[0275] Example 6 - Mitochondrial Dysfunction in Aged iNeurons

[0276] Mitochondrial dysfunction is a well-established hallmark of aging, manifested by decreased oxygen consumption. Based on data showing changes in neurite formation and select features of aging, mitochondrial activity in hiNeurons exposed to progerin was investigated using the Seahorse Mito Stress Test. As with physiological aging, neurons exposed to progerin were found to exhibit decreased basal and peak oxygen consumption in both pre- and post-aging ( Fig.12 AD). To exclude the possibility that a large number of cells died, leading to a decrease in the oxygen consumption rate (OCR), the viability of the cells was tested, confirming that the majority of cells were alive ( Fig.12 E).

[0277] Example 7 - Electrophysiological Recordings in Aged iNeurons

[0278] To test the functionality of aged iNs based on their electrophysiological properties, the cells were placed on multi-electrode arrays with and without rodent astrocytes. At D14 after induction, aged iNs showed reduced activity compared to non-aged iNs. However, by D21, the activity of the aged group returned to normal and showed burst durations similar to those of the non-aged group ( Fig.12 FH). This suggests that neuronal network communication is indeed affected in the early stages of maturation but is able to reach normal levels in later stages.

[0279] Example 8 - Transcriptome profiles of aged hiPSCs and aged iNeurons

[0280] Next, we investigated the transcriptome profiles of aged iPSCs and iNs by performing bulk RNA sequencing (RNA-seq) analysis. NGN2 , iPSC N2P and iN NGN2 Principal component analysis (PCA) demonstrated tight clustering of the control non-aged iPSC group and iN group, while the aged group was sparse ( Fig.13 A). Surprisingly, control iPSC N2P With aging iPSC N2P Together, this raises the concern that the cumate-inducible system may be leaky. To test this, we analyzed the number of reads for progerin and GFP from the transcriptional data. We found that the cumate-inducible system is indeed leaky, even though cells were not treated with cumate, control iPSCs N2P The expression of GFP-progerin was also shown ( Fig.14 Next, RNA-seq data were compared between the different groups to identify differentially expressed genes (DEGs). This enabled the discovery of transcriptome changes and the identification of unique and common genes associated with aging between the test groups. This comparison identified 2542 DEGs in aged iN at D14, of which 84 were age-related ( Fig.13 B). For aged iN at D21, the number of identified DEGs was almost twice as high, 5011, of which 122 were age-related ( Fig.13 B). Among the age-related genes, most were upregulated: 79 and 110 at D14 and D21, respectively. For aged iPSCs, 25 age-related DEGs were detected, out of a total of 1031 DEGs, including Nanog and OCT4 (POU5F1), which were both downregulated after progerin overexpression ( Fig.15To further support the aging phenotype, gene ontology (GO) terms were investigated. It was found that the upregulated DEGs in aged iNs at D14 and D21 were associated with DNA damage, apoptosis, and senescence GO terms, while the downregulated DEGs were associated with synapse, action potential, and neuronal development ( Fig.13 C and 16).

[0281] Example 9 - Epigenetic changes in aged hiPSCs and iNeurons

[0282] To date, this model system has been able to capture physiological aging phenotypes in both iPSCs and iNs. To test whether the epigenetic landscape of the cells was also affected by artificial aging, transcriptomic clock analysis was performed. These clocks are fitted algorithms that provide age estimates for cells based on the Tabula Muris Senis (mouse brain atlas) (Almanzar et al., 2020). These data show a slight shift from D14 to D21 in control non-aged iNs to a more aged phenotype. Notably, when progerin was induced, aging signaling was significantly affected, with a shift towards an older age at both D14 and D21 ( Fig.17 A). However, when trying to correlate the transcriptomic clock with the iPSC group, the cell age could not be predicted. Next, the data were analyzed based on a different epigenetic clock, the Horvath clock. This clock can predict cell age based on CpG methylation sites in DNA. Surprisingly, no significant changes in age were detected for aged iPSCs and iNs ( Fig.17 B) Using a modified form of this clock that links epigenetic changes to cell type, aged cells were found to be converting to CD4 T cell identity ( Fig.18 For the aging group, changes in in vitro cell morphology were also observed ( Fig.18 However, the transcriptome profile of the cells did not match that of CD4 T cells and still resembled that of neuronal cells ( Fig.18 ), suggesting that this may be due to different mechanisms. To further assess epigenetic changes, the Shannon entropy was measured based on the number of methylated CpG sites. This relatively new method can predict the chronological age of cells, because entropy increases with age, which also means that the epigenome loses information content (de Martin-Herranz Genome Biology 2019). In fact, it was found that both aged iPSCs and aged iNs had higher entropy than controls ( Fig.17 C) It should be noted that iPSC N2P and iN N2PThe controls also showed aging, which may be due to the previously observed leakiness in the cumate-inducible system.

[0283] Example 10 - Engineering and functional testing of CRISPR screening lines

[0284] To generate a progerin-based aging model for CRISPR screening, human iPSC lines were created that (i) could be artificially aged by overexpression of progerin and (ii) constitutively expressed the Cas9 protein ( Fig.19 A). Expression of the presenilin and Cas9 cassettes was confirmed by analysis of GFP (marking presenilin) ​​and mCherry (marking Cas9) activity, respectively. In the case of GFP overexpression (and thus the implicated presenilin), cells were treated with doxycycline for 5 days to ensure stable expression of the transgene. To determine a baseline for Cas9 expression and activity, engineered iPSC lines were transduced with a lentiviral vector encoding a gene targeting the β2 microglobulin (B2M) gene expressed on all nucleated cells.

[0285] The complex of Cas9 and sgRNA will disrupt the expression of B2M protein by introducing double-strand breaks in its genomic region, resulting in insertions or deletions (indels) and thus disrupting the open reading frame of B2M. Loss of B2M protein expression was observed after delivery of specific sgRNA compared to untreated controls, thus confirming Cas9 activity in this cell line ( Fig.19 A).

[0286] Conducted a large-scale CRISPR knockout (KO) screen targeting 1,000 genes potentially involved in aging and related processes (regeneration and senescence) Fig.19 BC). Of the 1000 candidate genes screened, 796 knockouts were identified in at least 20 cells. Some of the gene knockouts had an impact on cell fitness, thus confirming the functionality of the CRISPR / Cas9 and sgRNA architecture. This also allowed the identification of gene knockouts that affect the regeneration of iPSCs after progerin-induced aging at the transcriptional level.

[0287] Bulk RNA sequencing of the KOLF2-GFP-presenilin-Cas9 line was performed to decipher gene expression differences between iPSCs treated with doxycycline for 5 days to induce presenilin overexpression compared to untreated identical cells, thereby obtaining a transcriptome aging signature. In order to identify the effects of single gene knockouts on the aging signature of iPSCs at the single-cell level, linear discriminant analysis (LDA) was performed to create an “aging scale.” That is, the aging signature is used to generate a continuous variable (i.e., an aging score) that describes how “aged” a single cell is at the transcriptional level. This aging scale was integrated into a unified manifold approximation and projection (UMAP) map of all single cells treated in the CRISPR knockout screen ( Fig.19 D). This scale is used to evaluate the impact of knockout on aging / regeneration processes compared to cells transduced with a non-targeting sgRNA ("non-targeting") as a control.

[0288] Several knockouts were identified that had effects on aging / regeneration of iPSCs. These effects were either (i) pro-aging (i.e., knockout cells were restricted to a senescent state) or (ii) anti-aging (i.e., knockout cells ameliorated the effects of progerin on gene expression). In this example, knockout of EZH2 had an anti-aging effect in LDA, as cells carrying the corresponding knockout were less "senescent" compared to control conditions ( Fig.19 E). EZH2 has been implicated in cellular senescence, a process in which cells stop dividing and enter a state of permanent growth arrest, which is a hallmark of aging (Ito et al. (2018) Cell Reports). In addition, EZH2 has been reported to be part of the protein machinery that shapes the aging epigenome (Mozhui et al. (2017) Mech Ageing Dev). Together, these results suggest that the use of the CROP sequencing screening platform together with progerin-based aging models is an effective approach for pinpointing genes involved in aging and regeneration processes.

Claims

1. A method for transiently inducing aging in stem cells, the method comprising: (i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

2. The method according to claim 1, wherein the stem cell is a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a germ stem cell, a multipotent stem cell, an oligopotent stem cell, a unipotent stem cell or a tissue-resident stem cell.

3. A method for transiently inducing aging in somatic cells derived from induced pluripotent stem cells (iPSCs) by forward programming, the method comprising: (i) before or after forward programming of iPSCs, introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time, and (ii) removing or reducing one or more aging-inducing factors, thereby resulting in the reversal of one or more aging phenotypes in the cell.

4. The method according to claim 3, wherein the somatic cell is a neuron, a glial cell, a blood cell, an immune cell, a hepatocyte, a muscle cell, a myocyte, a fibroblast, a skin cell, a bone cell, a chondrocyte, an epithelial cell, an endothelial cell or an adipocyte.

5. The method according to any one of claims 1 to 4, wherein the introduction of one or more aging-inducing factors in step (i) comprises one or more expression cassettes comprising a sequence encoding the one or more aging-inducing factors, optionally wherein the expression cassette further comprises an inducible promoter regulated by a transcriptional regulatory protein.

6. The method according to claim 5, wherein the expression cassette and the gene encoding the transcriptional regulatory protein are inserted into a gene safe harbor site, The expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein.

7. The method according to claim 5, wherein the gene encoding the transcriptional regulatory protein is inserted into the first gene safe harbor site, and the expression cassette is inserted into the second gene safe harbor site, wherein the expression cassette further comprises an inducible promoter regulated by the transcriptional regulatory protein, and The first genetic safe harbor site and the second genetic safe harbor site are different.

8. The method according to any one of claims 5 to 7, wherein the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is controlled by an exogenously supplied substance such as tetracycline and its derivatives or cumate.

9. The method according to any one of claims 1 to 8, wherein removing or reducing one or more aging inducing factors in step (ii) comprises actively removing or reducing, thereby resulting in reversal of one or more aging phenotypes in the cell.

10. The method according to any one of claims 5 to 9, wherein the expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is reduced and / or inactivated by removing exogenously provided substances such as tetracycline and its derivatives or cumate.

11. A method according to any one of claims 1 to 10, wherein the one or more aging-inducing factors are alternatively spliced ​​forms of lamin A, such as presenilin, and / or wherein the one or more aging-inducing factors are mitochondrial DNA mutations, such as deletion of DNA polymerase subunit gamma (POLG), and / or wherein the one or more aging-inducing factors are aging-inducing compounds, such as telomerase inhibitors, reactive oxygen species (ROS), or compounds that damage or stress mitochondrial function, such as 6-hydroxydopamine (6-OHDA), valinomycin, CCCP, rotenone, or hydrogen peroxide, and / or One or more of the aging-inducing factors is an inducer of DNA damage, such as ionizing radiation, platinum-containing drugs, cyclophosphamide, chlorambucil or temozolomide. 12 . An artificial aged stem cell or a somatic cell derived from iPSC by forward programming, produced by the method of any one of claims 1 to 11 .

13. Use of the artificial aged stem cells according to claim 12 and / or somatic cells derived from iPSCs by forward programming in a method for identifying genes or gene combinations involved in reversal of an aged phenotype or maintenance of a non-aged phenotype in cells.

14. A method for identifying a gene or gene combination in a cell that is involved in reversing an aging phenotype or maintaining a non-aging phenotype, the method comprising the following steps: i) introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time; ii) optionally performing loss-of-function, inhibition, knockout, gain-of-function or a combination screening in said cell; iii) measuring the senescence phenotype of the cells; as well as iv) identifying genes or gene combinations as involved in reversal of an aged phenotype or maintenance of a non-aged phenotype when, upon loss-of-function, inhibition, knockout, gain-of-function or combination screening, the aged phenotype is altered and / or its progression is altered in cells compared to when the cells have not been screened.

15. The method according to claim 14, wherein in step (i), introducing one or more aging-inducing factors into the cells or exposing the cells to one or more exogenous aging-inducing factors for a period of time comprises introducing one or more expression cassettes into the cells, wherein the expression cassettes comprise sequences encoding the one or more aging-inducing factors and an inducible promoter regulated by a transcriptional regulatory protein, The expression of the sequence encoding the aging-inducing factor or the activity of the transcriptional regulatory protein is controlled by exogenously provided substances such as tetracycline and its derivatives or cumate.

16. A method according to claim 14 or claim 15, wherein the loss-of-function, inhibition, knockout, gain-of-function or combined screening comprises a genome-wide screening, and / or CRISPR is used for loss-of-function, inhibition, knockout, gain-of-function or combined screening.

17. The method according to claim 16, wherein the screen is a loss-of-function or inhibition screen and the CRISPR is CRISPRi, a knockout screen and the CRISPR is CRISPR-ko, a gain-of-function screen and the CRISPR is CRISPRa, or a combination screen and the CRISPR is a combination of two or more of: CRISPRi, CRISPR-ko and CRISPRa.

18. The use according to claim 13 or the method according to any one of claims 14 to 17, wherein the aging phenotype of the cell is selected from one or more of the following: proliferation, senescence, changes in cell and / or nuclear morphology, mitochondrial function, changes in gene expression, upregulation of lamin A and / or lamin C nuclear proteins, epigenetic marks associated with aging, changes in DNA or histone methylation, methylation entropy, DNA double strand breaks, telomere length, and transcriptome and / or epigenetic clocks, in particular transcriptome clocks, such as transcriptome features associated with an aging phenotype.

19. The use or method according to claim 18, wherein the ageing phenotype is decreased proliferation and / or increased senescence, and / or The changes in nuclear morphology are abnormal folding, blebbing, and / or loss of nuclear circularity, and / or wherein the mitochondrial function is reduced oxygen consumption and / or increased mitochondrial reactive oxygen species (ROS), and / or wherein the changes in gene expression are selected from one or more of the following: downregulation of somatic lineage identity genes, downregulation of mitochondrial genes, upregulation of apoptosis and / or aging-related genes, and upregulation of DNA damage-related genes, and / or wherein the epigenetic markers associated with aging are selected from one or more of the following: reduced heterochromatin trimethylation H3K9 (H3K9me3), reduced HP1γ and increased γH2AX, and / or where telomere length is shortened, and / or The transcriptome clock and / or epigenetic clock is a single-cell transcriptome clock and / or epigenetic clock, in particular a single-cell transcriptome clock, such as a transcriptome signature associated with an aging phenotype.

20. The use or method according to any one of claims 13 to 19, wherein the cell is a neuron derived from iPSC by forward programming, and optionally wherein the aging phenotype is a phenotype of a neurodegenerative disease, such as reduced neurite density and / or connectivity, shortened average dendrite length, shortened neurite diameter, downregulation of neuronal marker genes, or reduced electrophysiological activity.

21. A method of regenerating a somatic cell comprising altering the expression and / or activity of one or more genes identified by the use or method according to any one of claims 13 to 20.

22. The method according to claim 21, wherein the regenerated somatic cell comprises a reduced and / or slowed progression of an aging phenotype compared to a non-regenerated somatic cell, and wherein the aging phenotype is as defined in claim 18 or claim 19.

23. The method or use according to any one of claims 1 to 11 or claims 13 to 22, wherein the method or use is performed in vitro.

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