Ageing causes a decline in tissue regeneration owing to a loss of function of adult stem cell and progenitor cell populations1. One example is the deterioration of the regenerative capacity of the widespread and abundant population of central nervous system (CNS) multipotent stem cells known as oligodendrocyte progenitor cells (OPCs)2. A relatively overlooked potential source of this loss of function is the stem cell ‘niche’—a set of cell-extrinsic cues that include chemical and mechanical signals3,4. Here we show that the OPC microenvironment stiffens with age, and that this mechanical change is sufficient to cause age-related loss of function of OPCs. Using biological and synthetic scaffolds to mimic the stiffness of young brains, we find that isolated aged OPCs cultured on these scaffolds are molecularly and functionally rejuvenated. When we disrupt mechanical signalling, the proliferation and differentiation rates of OPCs are increased. We identify the mechanoresponsive ion channel PIEZO1 as a key mediator of OPC mechanical signalling. Inhibiting PIEZO1 overrides mechanical signals in vivo and allows OPCs to maintain activity in the ageing CNS. We also show that PIEZO1 is important in regulating cell number during CNS development. Thus we show that tissue stiffness is a crucial regulator of ageing in OPCs, and provide insights into how the function of adult stem and progenitor cells changes with age. Our findings could be important not only for the development of regenerative therapies, but also for understanding the ageing process itself.
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Raw and processed sequencing data have been deposited at the National Center for Biotechnology Information (NCBI) gene-expression omnibus (GEO) with accession number GSE133886 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133886). Source data include final quantifications from in vivo animal work.
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We thank D. Morrison for technical assistance and E. Paluch for helpful discussions and help with the manuscript. The work was supported by European Research Council (ERC) grant 772798 (to K.J.C.) and 772426 (to K.F.); the UK Multiple Sclerosis Society (to R.J.M.F.); Biotechnology and Biological Sciences Research Council (BBSRC) grant BB/M008827/1 (to K.J.C and R.J.M.F.) and BB/N006402/1 (to K.F.); the Adelson Medical Research Foundation (R.J.M.F. and D.H.R.); an EMBO Long-Term Fellowship ALTF 1263-2015 and European Commission FP7 actions LTFCOFUND2013, GA-2013-609409 (to I.P.W.); a Royal Society University Research Fellowship (to K.J.C.); and a core support grant from the Wellcome Trust and Medical Research Council (MRC) to the Wellcome Trust–MRC Cambridge Stem Cell Institute.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks D. Discher, M. Lutolf and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Representative flow-cytometry analysis of MACS-purified OPCs confirms that we are able to isolate a pure population of OLIG2+ NG2+ OPCs (indicated by percentages) from both neonatal and aged brains. NG2 is a marker specific for OPCs in the neonatal and adult CNS. b, EdU labelling of OPCs after 7 and 14 days in vitro. Scale bar, 25 µM. ‘GF’ indicates the growth factors FGF and PDGF. c, Quantifications of N = 3 replicates of neonatal and aOPCs in proliferation conditions on PDL-coated tissue-culture plastic after five days, showing that aOPCs proliferate poorly even in the presence of growth factors. d, Representative image of white matter labelled with OLIG2, EdU and CC1, from a 16-month-old female, showing that aOPCs also proliferate poorly in vivo. Scale bar, 50 µM. DAPI is used to stain nuclear DNA. e, Overview of the decellularization protocol. f, DAPI staining following the decellularization protocol shows no remaining nuclear DNA, indicating complete cell removal. Scale bar, 200 µM. g, Rat brains of different ages (neonatal or aged) were decellularized, fixed, and stained for chondroitin sulphate proteoglycans (CSPGs), showing that the ECM remains intact following decellularization. Scale bar, 20 µM. h, The recellularization protocol. OPCs (grey cells with tendrils) are MACS-purified using the OPC surface marker A2B5, then cultured for five days in proliferation conditions. A subset of the brain ECM cultures are fixed with paraformaldehyde, and the remainder are placed into differentiation conditions for a further five days. The coloured, connected circles represent the molecule paraformaldehyde. i, j, Representative images and a quantification of nOPCs seeded on neonatal and aged matrix, showing that nOPCs on aECM proliferate poorly. Panels a, d, f, g portray representative quantifications or images from N = 3 or more biological replicates.
Extended Data Fig. 2 OPCs grown in progressively stiff environments lose their proliferation and differentiation capacity.
a, Mean shear moduli determined by AFM of our fabricated ‘soft’ and ‘stiff’ hydrogels. b, c, MACS-purified nOPCs (stained for EdU and Sox10) cultured on stiff hydrogels lose their ability to proliferate following five days in proliferation conditions. nOPCs cultured on soft hydrogels, however, continue to proliferate. Scale bar, 40 µm. d, e, Similarly, nOPCs cultured on stiff hydrogels differentiate into oligodendrocytes (staining for OLIG2 and MBP) very inefficiently following five days in differentiation conditions. Conversely, nOPCs differentiated on soft hydrogels differentiate efficiently into oligodendrocytes. Scale bar, 100 µm. f, g, Representative images and quantifications from N = 3 replicates of EdU-labelled OPCs, seeded at 0.5×, 1× and 2× cell-seeding densities on increasingly stiff hydrogels, after 120 hours in culture show cell-density-independent, stiffness-dependent OPC activation. Scale bar, 100 µM. h, i, Labelling and quantifications from N = 3 viability assays of neonatal and aged OPCs on soft and stiff hydrogels after 48 hours in culture with propidium iodide (PI, a stain for DNA), showing that the stiffness effect does not affect cell death. The y-axis shows ratios of PI-stained cells to total cells. In all Extended Data Figures, averages represent means of biological replicates; error bars represent standard deviations; and P values are calculated by one-way ANOVA.
a–c, Gene-set-enrichment analysis (GSEA) reveals a number of ageing-related pathways that are differentially regulated between cultured aOPCs (a) and nOPCs (c) grown on soft hydrogels versus those grown on stiff hydrogels, and between aged and neonatal freshly harvested OPCs (b). HIF-1, hypoxia-inducible factor-1; mTOR, mammalian target of rapamycin; NOD, nucleotide-binding oligomerization domain; STAT, signal transducer and activator of transcription; TGF-β, transforming growth factor-β; TNF, tumour necrosis factor. d, Volcano plot of genes that are differentially expressed between aOPCs cultured on soft versus stiff hydrogels. Red dots show the 1,300 significantly upregulated expressed genes as determined from N = 3 biological replicates per condition (P < 0.05). e, Heatmaps showing the log2 FPKM expression of the 25 genes with the highest fold increase in expression between N = 3 biological replicates of aOPCs cultured on soft versus stiff hydrogels (left), and of the 25 genes with the highest fold increase in expression between aOPCs cultured on soft versus stiff hydrogels (right). ECM-related genes such as Dab1, Acan and Plxnd1 were upregulated in aOPCs cultured on stiff hydrogels, while cell-cycle and DNA-repair genes such as Cdk1na and Sirt7, OPC-activation genes such as Etv1, and Hippo-pathway genes such as Rassf2 were amongst the most upregulated genes in aOPCs cultured on soft hydrogels. All genes shown are significantly differentially expressed with a P value of 0.05 or less. f, Venn diagram showing that similar gene sets to those enriched in neonates are also enriched in nOPCs grown on soft hydrogels. g, Specific genes involved in genomic and epigenomic stability and in the activation of OPCs are upregulated both in nOPCs and in aOPCs grown on soft hydrogels.
Extended Data Fig. 4 Small molecules modulating the cell cytoskeleton promote the proliferation of aOPCs.
a, Using the 96-well-plate format, the GE Incell 2000 and a cell profiler for quantification, we optimized the dosing and timing of the small molecules Y27632 (‘Y27’) and blebbistatin (‘Bleb’) in order to identify the small-molecule conditions that maximize aOPC proliferation. Averages represent mean proportion of EdU+ OLIG2+ cells for N = 3 biological replicates. b, c, Representative images and quantifications from N = 3 biological replicates of the rates of proliferation of nOPCs cultured on soft and stiff hydrogels in the presence of 5 µm blebbistatin, showing that treatment with blebbistatin promotes the proliferation of OPCs similarly to soft hydrogels. Scale bar, 50 µm. d, e, Representative images and quantifications of N = 3 adult OPCs on soft hydrogels treated with blebbistatin or DMSO (as a control) show no change in the rates of proliferation, indicating that there is no stiffness-independent effect of blebbistatin. Scale bar, 50 µM. f, Box-and-whisker plots of AFM data from N = 3 aged vibratomed cortex treated with DMSO or 5 µM blebbistatin, indicating that the cortex softens significantly with blebbistatin treatment. The P value was calculated using a two-way Mann–Whitney test for two independent samples. g, h, Representative images and quantifications of EdU-labelled OLIG2+ CC1− OPCs seven days after the injection of DMSO or 5 µM blebbistatin into the grey matter of N = 3 14-month-old females. i–k, Overview and quantifications of the differentiation rates of aOPCs, following the injection of DMSO or 5 µM blebbistatin, at 14 days post-lesion in lesions induced by toxin (ethidium bromide) in vivo. The data are from N = 3 15-month-old age male rats. Differentiated oligodendrocytes are quantified as the proportion of CC1+ OLIG2+ cells per square millimetre of lesioned area. Scale bars, 50 µM. Source Data
Extended Data Fig. 5 The composition of the nuclear lamina of OPCs changes both with ageing and in response to niche stiffness.
a, qPCR of OPCs reveals a loss of Lmnb1 expression and gain of Lmna expression with ageing. Values represent averages of OPCs from N = 3 animals for each time point and are the log2 change in cycle threshold (∆CT) values normalized to the expression of Tbp, which is expressed in cells throughout the body. b, Representative images of in vivo cerebellar grey-matter cryosections from N = 3 biological replicates confirm changes in nuclear lamina that occur with ageing. Scale bar, 50 µm. White arrows highlight representative Ng2-expressing OPCs at each age. c, Representative western blot of Lamin B1 (LMNB1) and Lamin C (LMNC) proteins from freshly isolated OPCs of different ages confirms the qPCR data. ACTB is actin-β, a reference protein; p4 to p810 are protein fragments. Similar results were obtained for N = 3 biological replicates for each age group. d, RNA-sequencing data for nuclear Lmnb1 and Lmnb2 in neonatal and aged OPCs show low levels of expression of Lmnb2 in both age groups. e, f, Western blot quantifications of laminins from aged OPCs grown on soft and stiff hydrogels. g, Representative images from N = 3 biological replicates of changes in nuclear lamina in OPCs on hydrogels of different stiffnesses. Scale bar, 50 µM. h, Representative image from N = 3 biological replicates of red fluorescent protein (RFP)-conjugated non-targeting siRNA shows a high efficiency of siRNA transfection in vitro. Scale bar, 100 µM. i, qPCR on adult OPCs 48 hours after transfection with siRNAs, showing efficient knockdown of Lmna and Fak1 (also known as Ptk2) expression. Values represent averages of OPCs from N = 3 animals and are the log2 ∆∆CT values normalized to Tbp. j, k, Representative images and quantifications of the proliferation of N = 3 aOPCs in growth factors on stiff hydrogels following transfection with siRNAs for Lmna and Fak1. Scale bars, 50 µM. l, A representative image from N = 3 biological replicates of GFP-encoding mRNA in neonatal OPCs shows high-efficiency transfection. Scale bar, 100 µM. m, Representative image from N = 3 biological replicates, showing efficient transfection, high translation and proper protein localization of Lamin C in aOPCs. Scale bar, 25 µM. n, qPCR data five days post-transfection from RNA isolated from transfected OPCs. Means represent log2 ∆∆CT means across N = 2 biological replicates. o, p, Representative images and quantifications of N = 3 replicates in nOPCs on soft hydrogels show loss of proliferative capacity 120 hours after Lmnc mRNA overexpression but not after GFP overexpression. Scale bar, 100 µM.
Extended Data Fig. 6 PIEZO1, which mediates calcium flux, is highly expressed in OPCs but not other cells of oligodendrocyte lineage.
a, Representative images of aOPCs on soft and stiff hydrogels, showing that PIEZO1 is expressed in rat OPCs in vitro. Scale bar, 100 µm. b, Western blot for PIEZO1 in acutely isolated OPCs shows a modest increase in protein expression from neonates to adults. c, d, Representative images and quantifications of in situ hybridizations for Piezo1 and Pdgfra in aged mouse cortex using RNAScope, showing expression of Piezo1 in aged mouse OPCs. A negative control is included. Scale bar, 10 µm. e, t-distributed stochastic neighbour embedding (t-SNE) plots from a single-cell-sequencing study of human cells13 show that Pdgfra/Olig2 co-expressing OPCs from the adult CNS also highly express Piezo1 in adult white matter. f, g, Representative images and quantifications of aOPCs transfected with a control siRNA (‘Scramble’) or with a Piezo1 siRNA and placed in proliferation conditions for five days, indicating no stiffness-independent effect of PIEZO1. Scale bar, 100 µm. h, Representative Rhod-2-AM-stained live-cell images from N = 3 biological replicates of aOPCs on soft and stiff hydrogels transfected with siScramble or siPiezo1. i, Representative traces from N = 3 biological replicates of individual cells fluxing with calcium (∆Fintensity) over 270 seconds. Fluorescence was normalized to the maximum fluorescence intensity per cell over the acquisition time. j, Quantifications of the proportion of cells that fluxed calcium once or more throughout the 540-second image-acquisition period, showing that either seeding OPCs on soft hydrogels or overexpressing PIEZO1 inhibits the calcium flux. k, l, Representative images and proliferation quantifications from N = 3 biological replicates of aOPCs cultured in proliferation conditions for five days on a stiff hydrogel in the presence of 5 µM BAPTA (which chelates intracellular calcium), showing a boost in proliferation with calcium chelation. Scale bar, 100 µm.
a, Diagram showing CAS9-mediated genomic manipulation using in vitro transcribed (IVT) gRNA, Cas9 mRNA and in-house-made minicircle vectors overexpressing Piezo1-targeting shRNAs. This CAS9-mediated knock-in of shRNA–GFP has the benefit of producing a characterizable monotonic knockdown across the pool of cells expressing the GFP. b, qPCR data from RNA isolated from transfected OPCs, 48 hours after transfection with either the Piezo1 siRNA or the Piezo1 shRNA construct, both show an approximately 80% knockdown of Piezo1 mRNA. Means represent log2 ∆∆CT means from N = 3 biological replicates. c, Representative images from N = 3 biological replicates show high rates of co-transfection of the minicircle with Cas9 mRNA and IVT gRNA. Scale bar, 25 µM. d, e, PCR design and appropriate fragment length of the correctly knocked-in minicircle fragment construct, representative of results from N = 3 replicates. f, g, Representative images and quantifications show that CAS9-mediated knock-in of shPiezo1 fragments in aOPCs on stiff hydrogels phenocopies the effect of the Piezo1 siRNA in aOPCs. Scale bars, 100 µM. h, In order to knock down Piezo1 in endogenous OPCs in the aged mouse, we developed the strategy outlined here. A NHEJ-mediated knock-in inserts a construct into a gene specific to a given cell type. This construct contains a ribozyme-flanked second gRNA, targeting CAS9-mediated gene knockdown to a second locus. KO, knockout; PA, polyadenylation sequence. The rectangle and triangle represent the gRNA target sequence.
Extended Data Fig. 8 A nested CRISPR system efficiently labels OPCs with GFP and subsequently mutates the Piezo1 locus.
a, b, Representative images and quantifications of cells co-expressing GFP and OLIG2 across multiple regions of the CNS. Fewer than 5% of GFP-expressing cells express a marker other than OLIG2. Data taken from N = 3 control knockdown animals. Scale bars, 50 μm. c, RT–PCR shows Cas9 and GFP mRNA expression from whole-brain homogenate in N = 3 animals. d, A schematic and a DNA gel of N = 2 biological replicates of the Pdgfra locus following CRISPR/CAS9-mediated knock-in of a GFP transgene confirms construct knock-in in the correct position. KD, knockdown; NT, non-targeting gRNA (control construct); P1, forward primer for PCR; P2 reverse primer; ø symbol, uninjected control animal PCR. e, Outline of our experimental strategy to confirm the efficacy of our in vivo nested CRISPR approach. In brief, 5 × 1011 of each viral vector was tail-vein-injected into 18-month-old animals. At 35 days, brains were dissociated, and cells were FACS-sorted on the basis of their expression of Pdgfra and Olig2. Confirmatory PCR was carried out, as well as a surveyor assay to detect off-target small insertions and deletions (indels). f, A representative FACS plot from N = 3 biological replicates shows gating of sorted PDGFRA+ OLIG2+ OPCs from whole mouse brain. g, h, A representative agarose gel from a surveyor assay using T7 endonuclease I (g), and quantifications from N = 4 18-month-old animals (h), showing an indel rate of roughly 35% specific to FACS-sorted oligodendrocyte-lineage cells. i, Quantification of the off-target indels from N = 3 FACS-sorted OPCs from brains three weeks after AAV infection. N.D., not detected. j, k, qPCR results showing ∆∆CT from N = 3 biological replicates of PDGFRA/OLIG2-sorted cells from the in vivo infected aged CNS show a 75% reduction of Piezo1 indel-spanning mRNA and high expression of Piezo1 gRNA. l, Representative image from N = 3 biological replicates of GFP-plasmid electroporation in MEFs, showing a high efficiency of transfection. Scale bar, 50 µm. m–o, Schematic, western blot and quantifications of PIEZO1 protein levels in MEFs five days after electroporation of the Pdgfra-knock-in and Piezo1 gRNA construct. Quantifications represent averages from N = 3 replicate transfections for the PIEZO1 targeting and the non-targeting in vivo CRISPR constructs. Source Data
a, b, Representative images and box-and-whisker-plot quantification of white matter lesion (below the white line) stained only for OLIG2 show increased OPC infiltration into the lesion site following Piezo1 knockdown. c, d, Fluoromyelin staining and quantifications of the ratio of lesion area with positive fluoromyelin staining from N = 3 biological replicates show increased myelin deposition in Piezo1-knockdown animals. e, In order to show the role of PIEZO1 in development, we generated an additional in vivo CRISPR system. The diagram depicts GFP and a ribozyme-flanked Piezo1 gRNA under the control of the OPC-specific Cspg4 promoter. The square next to the triangle on the PIEZO1 genomic locus represents the gRNA target sequence. Scale bars, 100 µm. Source Data
Extended Data Fig. 10 A cell-type-specific CRISPR-mediated knockout of Piezo1 in OPCs during development increases both OPC proliferation and total cell number.
a, Outline of our experimental strategy to confirm the efficacy of our in vivo CRISPR approach. In brief, 5 × 1010 of each viral vector were tail-vein-injected into P1 pups. At 35 days, brains were dissociated, and cells were sorted by FACS on the basis of their expression of Pdgfra and Olig2. b, Representative FACS plot shows gating of a sorted PDGFRA+ OLIG2+ OPC population from whole mouse brain. c, d, An agarose gel from a surveyor assay using T7 endonuclease I (c), and quantifications from N = 3 P35 neonatal pups (d), showing a roughly 30% indel rate specific to oligodendrocyte-lineage cells. e, Representative images from N = 3 biological replicates, showing transgene specificity for OLIG2-expressing OPCs. f, qPCR results showing ∆∆CT from N = 3 biological replicates of PDGFRA+ OLIG2+ sorted cells from in vivo infected neonatal CNS, showing a roughly 55% reduction of Piezo1 indel-spanning mRNA relative to PDGFRA− OLIG2+ sorted cells. Representative lower-power images showing EdU labelling of PDGFRA/OLIG2-expressing cells in corpus callosum. Scale bar, 100 µm. g, Quantifications of PIEZO1 protein levels in MEFs five days after electroporation of the Piezo1 gRNA construct under the control of the Cspg4 promoter. Quantifications are from N = 3 biological replicates. h–j, Representative images and quantifications from N = 3 animals showing the total density of oligodendrocyte-lineage cells, as labelled by OLIG2/PDGFRA or OLIG2/CC1 co-expression, in P35 mouse corpus callosum following Piezo1 knockdown. Scale bars, 100 µm. Source Data
Supplementary Tables 1-11, Supplementary Notes containing additional discussion explaining the experimental methods chosen for the paper, highlighting sections of the manuscript that deserve extra attention, and images of uncropped DNA agarose and Western blot gels presented throughout the paper. The figure location for each gel is presented above the image panel and the perforated line indicates how the gel was cropped for the figure. The relevant DNA or protein ladder size is indicated on the side of the presented image.
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Segel, M., Neumann, B., Hill, M.F.E. et al. Niche stiffness underlies the ageing of central nervous system progenitor cells. Nature 573, 130–134 (2019). https://doi.org/10.1038/s41586-019-1484-9
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