- Record: found
- Abstract: found
- Article: found
Reprogramming progeria fibroblasts re‐establishes a normal epigenetic landscape
research-article
Author(s):
Zhaoyi Chen
1
,
2 ,
Wing Y. Chang
1 ,
Alton Etheridge
3 ,
Hilmar Strickfaden
4 ,
Zhigang Jin
4 ,
Gareth Palidwor
1
,
5 ,
Ji‐Hoon Cho
3 ,
Kai Wang
3 ,
Sarah Y. Kwon
1
,
6 ,
Carole Doré
1 ,
Angela Raymond
1 ,
Akitsu Hotta
7 ,
James Ellis
8
,
9 ,
Rita A. Kandel
10 ,
F. Jeffrey Dilworth
1
,
2
,
12 ,
Theodore J. Perkins
1
,
5
,
12 ,
Michael J. Hendzel
4 ,
David J. Galas
3 ,
William L. Stanford
1
,
2
,
6
,
11
,
12
,
Publication date (Electronic):
08 June 2017
Journal:
Aging Cell
Publisher:
John Wiley and Sons Inc.
Keywords:
aging,
epigenetics,
induced pluripotent stem cells,
Lamin A,
lamina,
progeria,
reprogramming
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Summary
Ideally, disease modeling using patient‐derived induced pluripotent stem cells ( iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson–Gilford progeria syndrome ( HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome‐wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient‐derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages.
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Most cited references35
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- Abstract: found
- Article: not found
Phenotype and course of Hutchinson-Gilford progeria syndrome.
Melissa A Merideth, Leslie B Gordon, Sarah Clauss … (2008)
Hutchinson-Gilford progeria syndrome is a rare, sporadic, autosomal dominant syndrome that involves premature aging, generally leading to death at approximately 13 years of age due to myocardial infarction or stroke. The genetic basis of most cases of this syndrome is a change from glycine GGC to glycine GGT in codon 608 of the lamin A (LMNA) gene, which activates a cryptic splice donor site to produce abnormal lamin A; this disrupts the nuclear membrane and alters transcription. We enrolled 15 children between 1 and 17 years of age, representing nearly half of the world's known patients with Hutchinson-Gilford progeria syndrome, in a comprehensive clinical protocol between February 2005 and May 2006. Clinical investigations confirmed sclerotic skin, joint contractures, bone abnormalities, alopecia, and growth impairment in all 15 patients; cardiovascular and central nervous system sequelae were also documented. Previously unrecognized findings included prolonged prothrombin times, elevated platelet counts and serum phosphorus levels, measured reductions in joint range of motion, low-frequency conductive hearing loss, and functional oral deficits. Growth impairment was not related to inadequate nutrition, insulin unresponsiveness, or growth hormone deficiency. Growth hormone treatment in a few patients increased height growth by 10% and weight growth by 50%. Cardiovascular studies revealed diminishing vascular function with age, including elevated blood pressure, reduced vascular compliance, decreased ankle-brachial indexes, and adventitial thickening. Establishing the detailed phenotype of Hutchinson-Gilford progeria syndrome is important because advances in understanding this syndrome may offer insight into normal aging. Abnormal lamin A (progerin) appears to accumulate with aging in normal cells. (ClinicalTrials.gov number, NCT00094393.) Copyright 2008 Massachusetts Medical Society.
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- Abstract: found
- Article: not found
Recapitulation of premature aging with iPSCs from Hutchinson-Gilford progeria syndrome
Hui Liu, Basam Barkho, Sergio Ruiz … (2011)
Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal human premature aging disease1–5, characterized by premature arteriosclerosis and degeneration of vascular smooth muscle cells (SMCs)6–8. HGPS is caused by a single-point mutation in the LMNA gene, resulting in the generation of progerin, a truncated splicing mutant of lamin A. Accumulation of progerin leads to various aging-associated nuclear defects including disorganization of nuclear lamina and loss of heterochromatin9–12. Here, we report the generation of induced pluripotent stem cells (iPSCs) from fibroblasts obtained from patients with HGPS. HGPS-iPSCs show absence of progerin, and more importantly, lack the nuclear envelope and epigenetic alterations normally associated with premature aging. Upon differentiation of HGPS-iPSCs, progerin and its aging-associated phenotypic consequences are restored. Specifically, directed differentiation of HGPS-iPSCs to SMCs leads to the appearance of premature senescence phenotypes associated with vascular aging. Additionally, our studies identify DNA-dependent protein kinase catalytic subunit (DNAPKcs) as a downstream target of progerin. The absence of nuclear DNAPK holoenzyme correlates with premature as well as physiological aging. Since progerin also accumulates during physiological aging6,12,13, our results provide an in vitro iPSC-based model to study the pathogenesis of human premature and physiological vascular aging. Three HGPS primary fibroblast lines, originally isolated from patients with the classical LMNA mutation (Gly608Gly), were transduced with retroviruses encoding OCT4, SOX2, KLF4, c-MYC, and GFP. Nanog-positive colonies were effectively obtained when using early passage, but not late passage (>25), HGPS fibroblasts (Fig. S1a). We focused on iPSC lines of a well characterized HGPS fibroblast line, AG019729–12. Compared to normal fibroblasts, HGPS fibroblasts (AG01972) showed abnormal nuclear morphology, reduced expression of the lamina components lamin B1 and LAP2β, loss of heterochromatin markers H3K9Me3, HP1α, and HDAC1, and reduced expression of nuclear proliferation marker Ki67 (Fig. 1a, S2). From HGPS fibroblasts, we derived six iPSC lines. In addition, we generated control iPSC lines from wild type fibroblasts (BJ and IMR-90 cell lines). Control and HGPS iPSC lines demonstrated pluripotent gene expression, demethylation of the OCT4 promoter and transgene silencing (Fig. 1b, S1, S3, S4a, and data not shown). They were maintained for more than 50 passages without a loss of pluripotency or the acquisition of detectable morphological or growth abnormalities. The pluripotency of each iPSC line was assessed by differentiation into the three embryonic germ layers in vitro, using embryoid body (EB) formation, and/or in vivo, by teratoma formation (Fig. S5a–d). Out of these lines, we primarily focused on HGPS-iPSC#4 and BJ-iPSC#3 for our studies (hereafter referred to as HGPS-iPSC or BJ-iPSC). Both BJ-iPSCs and HGPS-iPSCs were able to differentiate towards specialized mesoderm-derivatives such as smooth muscle cells (SMC), endothelial cells (Fig. S6a), or beating cardiomyocytes (Movie S1–S2). Moreover, all analyzed iPSC lines showed normal chromosomal integrity (Fig. S5e). Finally, LMNA sequencing confirmed the presence of the classical mutation in HGPS-iPSCs (Fig. S5f). Altogether, these data indicate that the somatic cells from HGPS patients, despite their significant premature senescence phenotypes and nuclear defects, have been properly reprogrammed and can be effectively maintained in a pluripotent state. Lamin A/C protein is expressed in differentiated somatic cells but is absent in embryonic stem cells (ESCs)11,14. Therefore, we next examined the expression of lamin A/C in the generated iPSC lines. As shown in Fig 2a, lamin A/C expression is significantly downregulated in iPSCs, compared to their parental fibroblasts, whereas lamin B1 transcripts were upregulated. Although progerin should follow a similar pattern of expression as observed for lamin A/C, LMNA expression is independent of promoter methylation status (Fig. S4b)15. Indeed, a complete loss of progerin mRNA in HGPS-iPSCs was observed (Fig. 2a). Furthermore, expression of lamin A/C and progerin proteins was practically undetectable (Fig. 2b, S4c). Since HGPS-iPSCs did not express progerin, we examined whether the nuclear abnormalities observed in HGPS fibroblasts would also be absent at the pluripotent stage. Our results indicate that all of the epigenetic, nuclear lamina and proliferation parameters analyzed in HGPS-iPSCs were indistinguishable from BJ-iPSCs (Fig. 2b–c, S3, S7). In addition, the nuclei of HGPS-iPSCs displayed the characteristic wrinkles and lobes observed in hESCs and iPSCs (Fig. S8), indicative of a reprogramming of the nuclear envelope components. Since the nuclear envelope associates with and regulates heterochromatin11,16, we next examined genome-wide CpG methylation in HGPS fibroblasts, BJ fibroblasts, HGPS-iPSCs, BJ-iPSCs, and H9 hESCs. Using bisulfite padlock probes and Illumina sequencing, we captured and quantified the methylation level of an average of 95,932 CpG sites within a set of 16,206 well annotated Differentially Methylated Regions (DMRs)17 per cell line (Table S1). The correlation coefficient of the global methylation levels between the pluripotent lines (BJ-iPSCs, HGPS-iPSCs and H9 hESCs) and the corresponding fibroblasts indicated that the generated pluripotent lines are much more closely related to each other and to hESCs than the two fibroblast lines (Fig. 2d). Interestingly, 586 autosome genes were found to be associated with regions that exhibited methylation differences between HGPS and BJ fibroblasts (Table S2, Fig S9a). Furthermore, based on DAVID18,19 analysis, we found that these genes were enriched for 21 Gene Ontology terms, most of which were related to development and transcriptional regulation (Fig S9a). In contrast, methylation differences between HGPS-iPSCs and BJ-iPSCs were only found for 33 autosome genes (Table S3), which showed no significant functional enrichment. Therefore, the presence of progerin in HGPS fibroblasts appears to lead to major epigenomic changes in various pathways. These changes were no longer present in HGPS-iPSCs, coinciding with the downregulation of progerin. Finally, genome-wide mRNA profiling demonstrated that HGPS-iPSCs and BJ-iPSCs are closely related together with H9 hESCs, and discrete from their parental fibroblasts (Fig. S9b–d). These results demonstrate the complete resetting of the nuclear architecture, epigenome and global gene expression in HGPS cells after being reprogrammed to pluripotency. To test whether the expression of progerin could be re-activated, we first subjected HGPS-iPSCs to in vitro differentiation via EB formation. Progerin mRNA was selectively induced in differentiated HGPS-iPSCs, but not in differentiated BJ-iPSCs (Fig. S10a). In contrast, lamin A was upregulated in both HGPS-iPSCs and BJ-iPSCs (Fig. S10a). This reversible suppression of progerin expression by reprogramming, and subsequent reactivation upon differentiation, provides a unique model system to study human premature aging pathologies. Progerin is known to accumulate mainly in arterial SMCs of HGPS patients, and vascular SMC degeneration is one of the characteristics of HGPS-associated arteriosclerosis6,7,20. In fact, vascular SMC senescence has been involved in the advanced arteriosclerosis of normal populations7,21,22. We therefore next asked whether SMCs differentiated from HGPS-iPSCs exhibit premature senescence phenotypes. Using a directed differentiation protocol, we obtained SMC populations from HGPS-iPSCs and BJ-iPSCs, the majority of which expressed characteristic SMC markers such as smooth muscle actin (SMA) and calponin (Fig. S6a). Immunoblotting and RT-PCR analyses confirmed the expression of progerin in HGPS-iPSC, but not BJ-iPSC-derived SMCs (hereafter referred to as “HGPS-SMC” and “BJ-SMC”, Fig. S6b–c). To model SMC senescence in vitro, the differentiated SMCs were serially passaged in culture. As shown in Fig. 3a–c and S10b, an increasing frequency of misshapen nuclei and a loss of the heterochromatin mark H3K9Me3 were specifically observed in HGPS-SMCs after serial passaging. HGPS-SMCs at later passages (i.e. passage 5) showed the typical characteristics of premature senescence, including increased senescence-associated-β-Gal (SA-β-Gal) staining (Fig. 3d–e, S10c), reduced telomere length (Fig. 3f), reduced number of Ki67-positive cells (Fig. 3g, S10d), and compromised cell proliferation (Fig. 3h, S10e). We also found a selective upregulation of senescence-related transcripts in HGPS-SMCs (Fig. S10f). To test whether the observed HGPS-related cell phenotypes were specific to SMCs, we differentiated HGPS-iPSCs into fibroblasts and measured progerin-associated parameters. Progerin expression in HGPS-iPSC-derived fibroblasts was detectable as early as passage 5 (Fig. S11a). However, we were unable to detect a loss of lamina or heterochromatin markers before passage 10 (Fig. S11b–c). Nonetheless, these defects were present specifically in HGPS-iPSC-derived fibroblasts, in contrast to control iPSC-derived fibroblasts analyzed at similar passage (data not shown). Thus, even though direct comparison of SMCs and fibroblasts is not possible due to their different culture conditions, our observations demonstrate that mesoderm lineages derived from HGPS-iPSCs display a characteristic HGPS phenotype. We next investigated whether progerin accumulation is the direct cause of the accelerated cell senescence observed in HGPS-SMCs. To this end, we induced ectopic expression of progerin in human primary vascular SMCs. We found that introduction of progerin in wild type SMCs resulted in compromised cell proliferation and nuclear defects, as we had observed in HGPS-SMCs (Fig. 3i, S12). As a complementary approach, we transduced HGPS-iPSCs with a lentiviral vector expressing a progerin-specific shRNA23. The modified iPSCs showed normal karyotypes as well as normal expression of lamina/epigenetic and pluripotent markers (Fig. S13a–b). After EB-based differentiation, both the mRNA and protein levels of progerin, but not those of lamin A, were substantially downregulated in the progerin-shRNA “corrected” HGPS-iPSCs compared to control cells (Fig. 3j, S13c–d). We next differentiated these “progerin-free” HGPS-iPSCs into SMCs (Fig. S14a). A dramatic improvement in the proliferation capability, as well as a downregulation of senescence-related transcripts, was found in the SMCs differentiated from the corrected HGPS-iPSCs (Fig. 3k and S14b–c). Furthermore, transduction of progerin shRNA into early passage HGPS-iPSC-derived fibroblasts resulted in a clear restoration of nuclear morphology and heterochromatin markers after extended culture (Fig. S15). Taken together, these data identify progerin as the key factor underlying the premature senescence phenotypes of HGPS-iPSC-derived cells. Since phenotypic characteristics of premature aging were able to be recapitulated by directed differentiation of the HGPS-iPSCs, we next investigated whether this model could serve to identify novel senescence-related markers. By using a sensitive MudPIT proteomic approach24,25, we identified DNA-dependent protein kinase catalytic subunit (DNAPKcs) as a hitherto unknown binding partner of progerin (Table S4). DNAPK holoenzyme, comprising of DNAPKcs and its regulatory subunits Ku70/Ku80, is involved in various aging-related cellular events26,27, and DNAPKcs or Ku70/Ku80-deficient mice exhibit accelerated aging27,28. To further confirm the association of progerin with DNAPKcs, we performed co-immunoprecipitation (co-IP) experiments. As shown in Fig 4a, ectopically expressed progerin associated tightly with endogenous DNAPKcs. In contrast, lamin A showed weak interaction with DNAPKcs, whereas both progerin and lamin A exhibited similar binding to lamin B1. Neither progerin nor lamin A co-immunoprecipitated with endogenous WRN protein. Since most of the nuclear proteins in complex with lamin A are destabilized in HGPS cells9,29, we analyzed the status of DNAPKcs in primary HGPS fibroblasts. We observed decreased nuclear DNAPKcs in HGPS fibroblasts when compared to normal fibroblasts (Fig. 4b–c). In addition, the regulatory subunits Ku70/Ku80 were also downregulated in HGPS fibroblasts (Fig. S16a). Interestingly, we detected a complete restoration of DNAPKcs/Ku70/Ku80 expression in HGPS-iPSCs (Fig. 4b, 4d, S3, S16b), although a deficiency in the expression of these proteins reappeared after differentiation into SMCs (Fig. 4e). These observations suggest that the downregulation of DNAPKcs in HGPS cells is dependent on the accumulation of progerin in differentiated cells. In fact, ectopic expression of progerin in primary vascular SMCs diminished DNAPKcs/Ku80 protein expression (Fig. 4f, S17). We next tested whether inactivation of DNAPKcs could partially contribute to some of the phenotypes observed in HGPS-SMCs. We found that knockdown of DNAPKcs reduced the proliferation of primary vascular SMCs (Fig. S18a). Finally, we extended our results and found that progressive loss of DNAPKcs/Ku70/Ku80 also occurs in fibroblasts isolated from normally aging individuals (Fig. S18b). Overall, our data suggest that deficiency of the DNAPK holoenzyme may constitute a novel marker for premature as well as physiological aging. In summary, our results not only highlight the plasticity of the lamina-epigenetics axis, but also point to the fact that the altered structure of the nuclear envelope, as well as the epigenetic modifications that accumulate during physiological aging12 or under specific disease conditions1,11, can be restored to normalcy by reprogramming (Fig. S19). The gradual onset and complexity of aging has impeded progress in understanding the pathogenesis of aging-related cardiovascular disorders. Recently, striking similarities between normal aging-associated and HGPS-associated arteriosclerosis have been reported6,16. Indeed, the levels of progerin increase gradually during physiological aging6. Our study provides the first evidence that, in a progerin-dependent manner, HGPS-iPSC-derived SMCs reach senescence-related phenotypes earlier than their normal counterparts. The iPSC-based accelerated aging model presented here and by Zhang et al30 may provide an avenue to model and study the pathogenesis of human aging-related vascular diseases as well as various human laminopathies1. METHODS SUMMARY iPSCs were generated from human fibroblasts with retroviruses encoding OCT4/SOX2/KLF4/c-MYC/GFP, and cultured on MEF feeder cells or Matrigel. SMCs were differentiated from iPSCs-derived CD34+ progenitor cells following an OP9-based protocol. ONLINE METHODS Cell culture H9 hESCs (WiCell Research) and iPSCs were maintained on a layer of mitotically inactivated mouse embryonic fibroblasts (MEFs) in hESC medium: DMEM/F12 (Invitrogen) supplemented with 0.1 mM non-essential amino acids (Invitrogen), 1 mM glutamax (Invitrogen), 20% Knockout Serum Replacement (Invitrogen), 55 μM β-mercaptoethanol (Invitrogen) and 10 ng/ml bFGF (Joint Protein hESCs and iPSCs were also cultured in Matrigel (BD Biosciences) with mTeSR medium (Stem Cell Technologies). Human HGPS fibroblasts AG01972, AG11498, AG06297, and normal fibroblasts GM00038 (9 year), AG05247 (87 year), and AG09602 (92 year) were purchased from Coriell Cell Repository. BJ normal human fibroblasts (CRL-2522) were purchased from ATCC. All human fibroblasts were cultured at 37°C in DMEM containing glutamax, non-essential amino acids, sodium pyruvate, and 15% fetal bovine serum (FBS). Human aortic smooth muscle cells were purchased from Lonza and maintained in SmGM-2 medium (Lonza, Cat. # CC-3182). Reagents Antibodies were obtained from the following sources. Abcam: anti-NANOG (ab21624), anti-H3K9Me3 (ab8898), anti-progerin (ab66587), anti-emerin (ab14208); anti-Ku70 (ab2172); Santa Cruz Biotechnology: anti-Oct-3/4 (sc-5279), anti-SOX2 (sc-17320), anti-HDAC1 (sc-7872), anti-DNAPKcs (sc-9051), anti-lamin A/C (sc-6215), anti-lamin A/C (sc-7293), anti-lamin B1 (sc-6217); Cell Signaling: anti-HP1α (2616); anti-Ku80 (2753); R&D systems: anti-Foxa2 (AF2400); Millipore: anti-TRA-1-60 (MAB4360) ; Sigma: anti-β-Tubulin III (T2200), anti-SMA(A5228), anti-Flag (M2), and anti-tubulin (T5168); Dako: anti-calponin (clone CALP); anti-endoglin (clone SN6h); BD Transduction Laboratories: anti-LAP2β (611000); MBL: Agarose-conjugated anti-GFP. Plasmids The pMXs vector containing the human cDNAs for OCT4, SOX2, KLF4 and c-MYC were purchased from Addgene (17217, 17218, 17219 and 17220, respectively). pBABE-puro-GFP-progerin and pBABE-puro-GFP-wt-lamin A were purchased from Addgene (17663 and 17662, respectively). Flag-progerin lentiviral vector was kindly provided by Dr. Lucio Comai 31. For the generation of the shRNA expression vectors against progerin and DNAPKcs, corresponding oligos (see Table S5) were cloned into a MluI/ClaI-cleaved pLVTHM plasmid (Addgene, 12247). All the constructs generated were subjected to DNA sequencing to confirm accurate shRNA target sequence. Retrovirus and lentivirus production For retrovirus production, 293T cells were transfected with the pMXs vectors carrying OCT4, SOX2, c-MYC, KLF4, or GFP cDNAs, together with the packaging plasmids (pCMV-gag-pol-PA and pCMV-VSVg, provided by Dr. Gerald Pao, The Salk Institute) using Lipofectamine 2000 (Invitrogen). Retroviruses were collected 36–48 h after transfection, and filtered through a 0.45 μM filter. Lentiviruses were generated by co-transfecting the pLVTHM vector together with the packaging plasmids (psPAX2 and pMD2.G, from Addgene, 12260 and 12259 respectively) into 293T cells using Lipofectamine 2000 (Invitrogen). Lentiviruses were collected 36 hours after transfection and concentrated by ultracentrifugation. iPSCs Generation For the generation of human iPSCs, human fibroblasts were seeded in a 6-well plate and spin-infected with a mix of high-quality retroviruses encoding OCT4, SOX2, KLF4, c-MYC, and GFP in the presence of 4 μg/ml polybrene. Three infections on consecutive days were performed. Six days after the first infection, fibroblasts were gently individualized with TrypLE (Invitrogen) and seeded onto fresh MEFs in the fibroblast culture medium. After 24 h, the medium was switched to hESC medium, and changed every 1–2 days depending on cell density. To establish the iPSC lines, colonies were manually picked and transferred onto MEF feeder cells for several passages before being transferred to Matrigel/mTesR conditions. Lentiviral infection of iPSCs HGPS-iPSC#4 cell line cultured on Matrigel was treated with 10 μM ROCK inhibitor Y-27632 for 1 h and then individualized with TrypLE. Cells were infected in suspension with either the concentrated lentivirus pLVTHM or pLVTHM-shRNA-Progerin in the presence of ROCK inhibitor and polybrene (4 μg/ml) for 1 h. Cells were centrifuged to remove the lentivirus and seeded back on fresh feeder MEFs in hESC media containing ROCK inhibitor. After being cultured for a few days, small colonies were manually passaged as a pool of colonies onto fresh MEFs to establish new iPSC lines. GFP expression was used as an indicator to determine successful integration of the lentiviruses. Cell differentiation For embryoid bodies (EBs) based differentiation, the iPSC colonies growing on MEFs were detached with dispase treatment, resuspended in DMEM/F12 medium supplemented with 0.1 mM non-essential amino acids, 0.5 mM L-glutamine, 10% FBS (Atlanta Biologicals), and 55 μM β-mercaptoethanol and cultured in low attachment 6-well plates for 4 days. The EBs were then plated on gelatin-coated plates and maintained for another 10–17 days. Differentiation of iPSCs into fibroblasts was performed as previously described32. Directed differentiation toward smooth muscle cells (SMCs) was performed essentially as previously described32 with slight modifications. Irritated OP9 cells were plated at 1 × 105 cells/well onto gelatinized 6-well plates in OP9 growth medium. After the formation of confluent iPS cell cultures for 4 and 5 days, undifferentiated iPS cells were harvested by treatment with 1 mg/mL dispase and dispersed by scraping to maintain the cells in small clumps. Concurrently, iPS cultures growing under the same conditions were used to obtain single cell suspension for counting. The iPSCs were added to OP9 cultures at a density of 3 × 105/2 mL per well of a 6-well plate in half TesR medium and half hESC media. iPSCs were allowed to recover for 1–2 days in hESC media. At day 0 of differentiation, the media was changed to Knockout DMEM supplemented with 10% FBS (HyClone), 10 mM β-mercaptoethanol, 1 mM L-glutamine, and 100 mM nonessential amino acids. The iPSC/OP9 cocultures were incubated for up to 10 days at 37°C in 5% CO2 conditions with medium change every other day. After 10 days of differentiation, the coculture cells were harvested with TrypLE (Invitrogen) for single-cell suspension and labeled with CD34 microbeads kit (Miltenyi Biotech, Cat. #130-046-702). Following the manufacturer's protocol, cells were passed through MS separation column attached to a Midi-MACS separation unit (Miltenyi Biotech) to obtain a magnet-retained fraction of purified CD34+ cells. Isolated CD34+ cells were then plated in smooth muscle cell media (SmGM-2 BulletKit, Lonza, Cat. # CC-3182) and maintained at 37°C in 5% CO2 conditions with medium change every 2–3 days34. SMCs were passaged using TrypLE (diluted 1:4) for 3 minutes at 37°C. To analyze early onset of senescence, cells were passaged at a ratio of 1:3 (~6000–7500 cells/cm2) only when the cells reached confluence. To calculate population doublings, SMCs seeded at 3500 cells/cm2 were passaged once culture reached 85–90% confluence. Cell growth was measured at every passage by calculation of accumulated population doublings using the formula (logH -logS) / log2.0 (H=number of cells harvested; S=number of cells seeded on the first day of each passage). Protein and mRNA analysis Cells were lysed and subjected to immunoblotting analysis according to the previously described method35. Total RNA was extracted using Trizol (Invitrogen) followed by cDNA synthesis using High capability RNA-to-cDNA Mater Mix (Invitrogen). Quantitative RT-PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). Primer sequences are listed in Table S5. Immunofluorescence microscopy Cells were fixed with 4% formaldehyde in PBS at room temperature (RT) for 20–30 min. After fixation, cells were treated with 0.4% Triton X-100 in PBS for 5 min at RT. After blocked with 10% FBS in PBS for 30 min, cells were incubated at RT for 1 h or at 4°C overnight with the primary antibody, followed by washing in PBS and incubation at RT for 1 h with the corresponding secondary antibody. Nuclei were stained with Hoechst 33342 (Invitrogen). Quantitative microscopy measurements were carried out as described previously9. Error bars represent standard deviations. Immunohistochemical detection of NANOG Cells were fixed with 4% formaldehyde in PBS at RT for 30 min, and permeabilized with 0.4% Triton-X100 in PBS for 10 min. Then the cells were incubated overnight with rabbit anti-human NANOG antibody in 1% BSA/PBS, followed by incubation with a secondary biotin-conjugated anti-rabbit antibody for 2 hours. Finally, cells were incubated with streptavidin-HRP for 1 hour (Vector), and NANOG-positive cells were visualized with a DAB substrate kit (Vector). Teratoma analysis To test pluripotency in vivo, NOD-SCID IL2Rgammanull mice (Jackson laboratories) were injected with the indicated iPSC lines and teratoma formation assessed. Briefly, ~106 iPSCs in ~50uL of hESC medium were injected into the testis or kidney capsule of anesthetized mice. Mice were monitored for teratoma formation and euthanized ~6–12 weeks after injection. Teratomas were harvested, processed and analyzed by hematoxylin-eosin staining and immunostaining. All animal experiments were performed with approval of The Salk Institute Institutional Animal Care and Use Committee (IACUC). Mutation validation Primer sequences to amplify exon 11 of the LMNA gene are listed in Table S5. 50 μl PCR reactions using 3 ng genomic DNA templates, 100 nM of the forward and reverse primers with 25 μl Taq 2× Master Mix (NEB) was performed at 94°C for 2 min, 34 cycles of 94°C 30 s, 55.5°C for 40 s, and 72°C for 40 s, and finally 72°C for 3 min. Products were purified with 0.9× volume of AMPure beads (Agencourt). Amplicons were sequenced by capillary Sanger sequencing (Genewiz). Results were visualized using an ABI Sequence Scanner. Genome–wide DNA methylation analysis Genomic DNA was extracted using ALLPrep DNA/RNA Mini kit (Qiagen). Bisulfite conversion and capture reaction was carried out on each sample (genomic DNA of fibroblasts, iPSCs, or hESCs). The detailed protocol for genomic DNA methylation has been described previously36, and the detailed information for DNA methylation is presented in Table S1. Bisufite sequencing of OCT4 and LMNA promoters Bisulfite conversion was carried out using 2 μg of purified genomic DNA using the Zymo EZ-DNA Methylation Gold Kit (Zymo Research) following the manufacturer's instructions. PCR was set up using previously published primers15. Cycling was terminated at 35 cycles. PCR products were purified using 2 % Size-Select E-gel (Invitrogen) and reamplified for 10 cycles using Phusion HF enzyme (NEB). PCR products were cloned using Zero-blunt PCR Cloning kit (Invitrogen) and heat transfected to TOP10 E. coli competent cells (Invitrogen). Individual colonies were selected and sent for single pass sequencing. DNA microarray and Bioinformatics analysis The GeneChip microarray processing was performed by the Functional Genomica Core in the Institute for Research in Biomedicine (Barcelona, Spain) according to the manufacturer's protocols (Affymetrix, Santa Clara, CA). The amplification and labeling were processed as indicated in Nugen protocol with 25ng starting RNA. For each sample, 3.75μg ssDNA were labeled and hybridized to the Affymetrix HG-U133 Plus 2.0 chips. Expression signals were scanned on an Affymetrix GeneChip Scanner (7G upgrade). The data extraction was done by the Affymetrix GCOS software v.1.4. The statistical analysis of the data was performed using ArrayStar 3. Briefly, raw CEL files were imported together with gene annotation from NetAffix (from 11/13/2009) and after checking for top replication quality for each of the 5 pairs of samples (R2>0.99), data was summarized at the gene level (20,765 genes) and the median was used for each gene and sample type. As both H9 hESCs and HGPS-iPSCs originate from female samples, and in order to remove any possible bias introduced by the X and Y chromosome-coded genes, we performed the same analysis with only autosome genes (19,884 genes). The result of the hierarchical clustering is very similar to the one using all genes and is shown in Figure 2f. In addition, a principal component analysis was performed on RMA-normalized probeset intensity values for autosomes using the prcomp function in R (http://www.r-project.org/) (the same figure including all genes gave highly similar results, data not shown). A figure illustrating the two first principal components is shown in figure S9c. Differences between some of the samples is shown using scatter plot of RMA-normalized intensity values in figure S9b. Multidimensional Protein Identification Technology (MudPIT) analysis of progerin-associated proteins The immunoprecipitation for MudPIT assay was performed as previously described24,25. In brief, HEK293T cells were transfected with GFP-progerin or GFP and maintained in culture for 48 h. After cells were lysed, the GFP-progerin, GFP, and their associated proteins were immunoadsorbed to anti-GFP agarose. The immunoprecipitates were then eluted with 8 M urea in 100 mM Tris, pH 8.5. The samples were reduced by adding 0.3 μL of 1M TCEP (for a final concentration of 5 mM TCEP) and incubated at RT. To alkylate, 1.2 μL of Iodoacetamide (10 mM final concentration) was added and the samples were subsequently incubated at RT in the dark for 15 minutes. The addition of 180 μL of 100 mM Tris pH 8.5 diluted the solutions to 2 M Urea. Calcium chloride (100 mM) was then added (2.4 μL) for a final concentration of 1 mM CaCl2. Trypsin (0.5 μg/μL) was added in the amount of 7.0 μL. The resulting mixtures were then shaken for 18 hours and incubated in the dark at 37 °C. To neutralize 13.5 μL of Formic Acid (90%) was added for a final concentration of 5% Formic Acid. The tubes were centrifuged for 30 minutes at 2 °C in a table-top centrifuge. Upon completion of the digestion, the proteins were pressure-loaded onto a fused silica capillary desalting column containing 3 cm of 5-μm strong cation exchange (SCX) followed by 3 cm of 5-μm C18 (reverse phase or RP material) packed into an undeactivated 250-μm i.d capillary. Using 1.5 mL of buffer A (95% water, 5% acetonitrile, and 0.1% formic acid) the desalting columns were washed overnight. Following the desalting process, a 100-μm i.d capillary consisting of a 10-μm laser pulled tip packed with 10 cm 3-μm Aqua C18 material (Phenomenex, Ventura, CA) was attached to the filter union (desalting column-filter union-analytical column) and the entire split-column (desalting column-filter union-analytical column) was placed in line with an Agilent 1100 quaternary HPLC (Palo Alto, CA) and analyzed using a modified 6-step separation, described previously25. The buffer solutions used were 5% acetonitrile/0.1% formic acid (buffer A), 80% acetonitrile/0.1% formic acid (buffer B), and 500 mM ammonium acetate/5% acetonitrile/0.1% formic acid (buffer C). Step 1 consisted of a 90 min gradient from 0–100% buffer B. Steps 2–5 had the following profile: 3 min of 100% buffer A, 2 min of X% buffer C, a 10 min gradient from 0–15% buffer B, and a 97 min gradient from 15–45% buffer B. The 2 min buffer C percentages (X) were 20, 40, 60, 80% respectively for the 6-step analysis. In the final step, the gradient contained: 3 min of 100% buffer A, 20 min of 100% buffer C, a 10 min gradient from 0–15% buffer B, and a 107 min gradient from 15–70% buffer B. As peptides eluted from the microcapillary column, they were electrosprayed directly into an LTQ 2-dimensional ion trap mass spectrometer (ThermoFinnigan, Palo Alto, CA) with the application of a distal 2.4 kV spray voltage. A cycle of one full-scan mass spectrum (400–1400 m/z) followed by 8 data-dependent MS/MS spectra at a 35% normalized collision energy was repeated continuously throughout each step of the multidimensional separation. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur datasystem. As each step was executed, its spectra were recorded to a RAW file. This data was then converted into .ms2 format through the use of RawXtract (Version 1.9). From the .ms2 files, poor quality spectra were removed from the dataset using an automated spectral quality assessment algorithm37. MS/MS spectra remaining after filtering were searched with the SEQUEST™ algorithm38 against the NCBI RefSeq Human (04-23-2010) protein database concatenated to a decoy database in which the sequence for each entry in the original database was reversed25,39. All searches were parallelized and performed on a Beowulf computer cluster consisting of 100 1.2 GHz Athlon CPUs40. No enzyme specificity was considered for any search. SEQUEST results were assembled and filtered using the DTASelect (version 2.0) program. DTASelect 2.0 uses a linear discriminant analysis to dynamically set XCorr and DeltaCN thresholds for the entire dataset to achieve a user-specified false positive rate. The false positive rates are estimated by the program from the number and quality of spectral matches to the decoy database. The hits detected uniquely in the GFP-progerin sample but not in GFP sample represent proteins that are specifically associated with progerin, by either direct or indirect interactions. Co-Immunoprecipitation BJ human fibroblasts were transduced with retrovirus encoding GFP-progerin, GFP-lamin A or GFP, and maintained in culture for 72h. For immunoprecipitation, cells were lysed in ice-cold lysis buffer (250 mM NaCl, 0.5% Triton X-100, 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% glycerol, and complete protease inhibitor cocktail (Roche Diagnostics)). Samples were briefly sonicated and immunoprecipitated by incubating with anti-GFP agarose. The immunoprecipitates were washed extensively in lysis buffer, eluted in SDS sample buffer, and subjected to immunoblotting. Senescence-associated beta-galactosidase (SA-βgal) assay SA-βgal assay were performed based as previously described methods41. Measurement of telomere length Genomic DNA was isolated from 1 × 106 cells. The telomere-specific oligonucleotide probe (5'-TTAGGGTTAGGGTTAGGGTTAGGG-3'; ValueGene) was end-labeled using γ-32P-ATP (MP Biomedicals) and T4 polynucleotide kinase (NEB). Two μg of genomic DNA for each sample was digested with AluI (NEB) and MboI (NEB) and subjected to Southern analysis with the telomere-specific probe. Mean telomere length was calculated from ΣODi/(ΣODi/ΣMWi). ODi and MWi are optical density and molecular weight at a given position i, respectively. Cell proliferation assay Cell proliferation was determined with CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium)), according to the protocol provided by the manufacturer (Promega). Statistical analysis Results are presented as mean±s.d. Comparisons were performed with student's t-test or one-way anova. p<0.05 was defined as statistically significant. Supplementary Material 1 2 5 6 7 8 9
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Genomic instability in laminopathy-based premature aging.
Baohua Liu, Jianming Wang, Kui Ming Chan … (2005)
Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.
Author and article information
Contributors
William L. Stanford:
ORCID: http://orcid.org/0000-0002-5813-8016
wstanford@ohri.ca
Journal
Journal ID (nlm-ta): Aging Cell
Journal ID (iso-abbrev): Aging Cell
Journal ID (doi): 10.1111/(ISSN)1474-9726
Journal ID (publisher-id): ACEL
Title:
Aging Cell
Publisher:
John Wiley and Sons Inc.
(Hoboken
)
ISSN
(Print):
1474-9718
ISSN
(Electronic):
1474-9726
Publication date
(Electronic):
08
June
2017
Publication date
(Print):
August
2017
Volume: 16
Issue: 4
(
doiID:
10.1111/acel.2017.16.issue-4
)
Pages: 870-887
Affiliations
[
1
]
The Sprott Centre for Stem Cell Research
Regenerative Medicine Program
Ottawa Hospital Research Institute
Ottawa
Ontario
Canada
K1H 8L6
[
4
]
Cross Cancer Institute and the Department of Experimental Oncology
Faculty of Medicine and Dentistry
University of Alberta
Edmonton
Alberta
Canada
T6G 1Z2
[
5
]
Ottawa Bioinformatics Core Facility
The Sprott Centre for Stem Cell Research
Ottawa Hospital Research Institute
Ottawa
Ontario
Canada
K1H 8L6
[
8
]
Program in Developmental and Stem Cell Biology
The Hospital for Sick Children
Toronto
Ontario
Canada
Author notes
[*]
[*
]
Correspondence
Dr. William L. Stanford, The Ottawa Hospital Research Institute, The Ottawa Hospital, 501 Smyth Rd, Box 511, Ottawa, Ontario, Canada K1H 8L6 . Tel.: 613‐737‐8899 Ext. 75495; fax: 613‐739‐6294; e‐mail: wstanford@ 123456ohri.ca
Dr. William L. Stanford, The Ottawa Hospital Research Institute, The Ottawa Hospital, 501 Smyth Rd, Box 511, Ottawa, Ontario, Canada K1H 8L6 . Tel.: 613‐737‐8899 Ext. 75495; fax: 613‐739‐6294; e‐mail: wstanford@ 123456ohri.ca
Author information
William L. Stanford
http://orcid.org/0000-0002-5813-8016
Article
Publisher ID: ACEL12621DOI: 10.1111/acel.12621
PMC ID: 5506428
PubMed ID: 28597562
SO-VID: dca69afe-8133-42c2-b103-69b021b2e370
Copyright © © 2017 The Authors.
Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
License:
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
History
Date
accepted
: 21
April
2017
Page count
Figures: 8,
Tables: 1,
Pages: 18,
Words: 11695
Funding
Funded by: Canadian Institutes of Health Research
Award ID: MOP‐133570
Funded by: Progeria Research Foundation
Award ID: PRF‐009‐003
Funded by: Heart & Stoke Foundation of Canada
Award ID: G‐13‐0003053
Funded by: Ontario Ministry of Research and Innovation
Funded by: Luxembourg‐ISB program
Funded by: Canadian Heart & Stroke Postdoctoral Fellowship
Funded by: Ontario Ministry of Research and Innovation Postdoctoral Fellowship
Funded by: Tier 1 Canada Research Chair in Integrative Stem Cell Biology
Categories
Subject:
Original Article
Subject:
Original Articles
Custom metadata
source-schema-version-number 2.0
component-id acel12621
cover-date August 2017
details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_NLMPMC version:5.1.4 mode:remove_FC converted:21.07.2017
ScienceOpen disciplines: Cell biology
Data availability:
ScienceOpen disciplines: Cell biology
Keywords: aging, epigenetics, induced pluripotent stem cells, lamin a, lamina, progeria, reprogramming