Cell strain-derived induced pluripotent stem cell as a genetically controlled approach to investigating aging mechanisms and viral pathogenesis

The expansion of the geographic footprint of dengue viruses (DENVs) and their mosquito vectors have affected more than half of the global population, including older adults who appear to show elevated risk of severe dengue. Despite this epidemiological trend, how age and senescence impact virus-host interactions involved in dengue pathogenesis to increase the risk of severe dengue is poorly understood. Herein, we show that conversion of diploid cells with finite lifespan into iPSCs followed by differentiation back into cell strain can be an approach to derive genetically identical cells at different stages of senescence to study virus and aging host interactions. Our findings show that cellular senescence impact the host response to infection and the ensuing outcome. We suggest iPSC-derive cell strains as a potentially useful technical approach to genetically controlled host-virus interaction studies to understand how aging impact viral pathogenesis.


INTRODUCTION 28
Dengue is the most common mosquito-borne viral disease globally (1). This acute 29 disease, which when severe can be life-threatening, is caused by four genetically 30 distinct dengue viruses (DENVs) (DENV1,-2,-3 and -4), all of which belong to 31 Flavivirus genus. An estimated 390 million infections occur annually (2) and 32 populations throughout the tropics face frequent and recurrent dengue epidemics. 33 More are expected to be affected as the geographic footprint of the Aedes 34 mosquitoes that transmit DENV expand from the tropical to the subtropical regions of 35 the world (3). 36 When frequent and recurrent dengue epidemics first emerged in Southeast Asia 37 after the Second World War, dengue was primarily a paediatric disease (4, 5). Early-38 life exposure to DENV remains enriched in children in certain parts of the region, 39 leading to immunity by early adulthood (5). However, changes in the urban 40 population demographics as well as vector distribution have led to a shift in the 41 burden of dengue to include adults and even the elderly (6-10). Dengue in older 42 adults present public health challenges as these individuals appear to experience 43 greater morbidity and mortality rates (11). Epidemiological observations have found 44 increased rates of hospital and intensive care admissions (11), length of 45 hospitalisation (12), and risk of severe dengue (12-15). Although age is associated 46 with increased prevalence of co-morbidities, such as cardiovascular diseases and 47 diabetes that also complicate dengue (16,17), age alone has also been shown to be 48 a risk factor for severe dengue (12). This age-related increased risk of severe 49 disease extends beyond dengue. Vaccination with the live attenuated yellow fever 50 vaccine (YF17D) in those above 60 years of age has, despite the attenuated nature 51 of YF17D, has been associated with severe viscerotropic infection and disease (18, 52 19). 53 Despite the increased risk of poor clinical outcome in older adults, how aging affects 54 the pathogenesis of DENV infection and severe adverse events following YF17D 55 vaccination has remained undefined. A major limitation is the lack of suitable in vitro 56 tools. Cell lines that are commonly used in virus-host interaction studies are immortal 57 and do not age. Cell strains, or diploid cells with finite lifespan, do age (20). 58 However, most of these cell strains were developed decades ago and are thus 59 mostly close to the end of their finite lifespan. Moreover, global stocks of several of 60 these cell strains are approaching depletion (21). Cell strains at a spectrum of 61 chronological ages are thus not readily available for virus and aging host interaction 62 studies. 63 64 Herein, we explored the use of induced pluripotent stem cells (iPSCs) generated 65 from senescent diploid cells, and then differentiated from iPSCs back into senescent 66 cells as a resource for age-dependent viral pathogenesis investigations; conversion 67 of diploid cells to iPSCs serve as a renewable resource for differentiation and 68 passaging into genetically identical cells at different stages of senescence. We show 69 that early passages of differentiated cells display markers of differentiation while later 70 passage cells exhibit cellular senescence. The difference in passage number 71 influences the flavivirus infection phenotype, potentially offering an in vitro system to 72 study host immune response to infection in the context of cellular senescence. 73

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Senescent cell strains can be reprogrammed into induced pluripotent stem 77 cells. Cell strains, WI-38 and MRC-5 were created as cancer free, virus free cells for 78 vaccine production (20,22). These diploid cell strains, however, have since proven 79 useful for in vitro cell biology and basic virology studies as they are not immortalised 80 (23,24). We reprogrammed these cell strains into iPSCs using non-modified of 81 Yamanaka factors (Klf4, Oct4, Sx2 and c-Myc) as well as the transcription factors 82 Nanog and Lin28 (25) rather than conventional dedifferentiating techniques such as 83 retro-or lentivirus vectors that alter the host cell genome (Figure 1). Fourteen days 84 post-transfection with the cocktail of reprogramming mRNA, three suspected iPSC 85 colonies were isolated from WI-38 (W1-3) and 6 colonies from MRC-5 cells (M1-6).  Colonies W1 and M3, were conveniently selected for further characterisation. We 106 found increased expression of iPSC cell surface marker TRA-1-60 and transcription 107 factor OCT 4 in these colonies (Figure 1c). Reprogramming from fibroblasts to iPSCs 108 was further confirmed at the level of transcription by significantly decreased 109 expression of fibroblast associated genes (acta2, col3a1, fsp, ltbp2, timp1 and vim) 110 and increased expression of iPSC gene markers (dmnt3b, htert, nanog, oct4, sox2 111 and tdgf1) relative to the parental fibroblasts (Figure 1d-e). 112 113 A hallmark of cell strains WI-38 and MRC-5 fibroblasts is that they undergo 114 senescence (20) due to lack of expression of human telomerase (hTERT) which 115 prevents telomere shortening. We thus measured hTERT expression in our colonies. 116 Expression of hTERT was upregulated in W1 and M3 iPSCs as compared to their 117 respective parental fibroblasts ( Figure 1e). 118

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Stemness was also validated through measuring alkaline phosphatase (AP) activity, 120 which is present in stem but not differentiated cells. Indeed, parental WI-38 121 fibroblasts stained negative for AP while W1 and M3 iPSCs demonstrated positive 122 pink staining of AP activity (Supplementary Figure S1b). 123

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To gain insights into the differences between passage 1 (P1) to P4 differentiated 151 cells, we analysed whole genome expression of these cells using microarray.    We have used a chemically defined media to differentiate the iPSCs into an adherent 333 cell monolayer, that was followed through serial passaging. We have used this 334 relatively straightforward approach as a proof-of-concept demonstration of WI-38 and 335 MRC-5 derived iPSCs and iPSC-derived differentiated cells as in vitro models to study 336 age-related effects on viral infection. Indeed, our transcriptional analysis showed that 337 the baseline expression of aging-related genes were increased upon passaging of W1 338 and M3 cells. To our knowledge, such an approach to derive isogenic cells of different 339 replicative ages for infection studies has not been previously attempted. Future studies 340 could make use of better defined differentiation protocols using well established kits. 341 Alternatively, iPSCs could also be differentiated through the use of transcriptions 342 factors computationally predicted by mogrify (35)

MATERIALS AND METHODS 381
Cells and culture conditions. Human diploid fibroblast WI-38 (female) and MRC-5 382 (male) cell strains were maintained in fibroblasts growth media (Minimum Essential 383 Media, 10% FCS, 1% GlutaMAX, 1% penicillin/streptomycin) at 37°C, 20% O2, 5% 384 CO2. Cell strains were passaged with TrypLEä Expression Enzyme. BHK21 cells used 385 for plaque assay were grown in RMPI Medium 1640 (Gibco), 2% FCS and 1% 386 penicillin/streptomycin at 37°C, 20% O2, 5% CO2. 387 388 All stem cells were cultivated on 1% Geltrexä coated cell culture ware in mTeSRä1 389 or TeSRä-E8ä media for maintenance. Stem cells were passaged according to 390 manufacturers' recommendations with ACCUTASEä or ReLeSRä where appropriate. 391 Briefly, spent media was removed from the stem cells and ReLeSRä was added and 392 incubated at room temperature for one minute. ReLeSRä was then removed, cells 393 were incubated at 37°C, 20% O2, 5% CO2 for 6:30min, fresh media was added gently, 394 and cells were re-suspended by tapping the plate for 1min. ACCUTASEä was added 395 to cells and incubated for 7min at 37°C, 20% O2, 5% CO2. Cells were resuspended, 396 transferred to a conical tube and spun at 250 x g for 5min at room temperature. The 397 ACCUTASEä was then removed and cells were re-suspended in desired media with 398 Immunofluorescent assay. Primary antibodies against iPSC markers TRA-1-60 433 (ab16288, Abcam â , 1/500) and OCT4 (ab181557, Abcam â , 1/250) were used for 434 immunofluorescence assays to determine the expression of stem cell proteins in 435 iPSCs or fibroblasts. Spent media was removed, cells were washed once with PBS 436 and subsequently dislodged using ACCUTASEä according to manufacturers' 437 instructions. The cells were spun down at 250 x g for 5min at room temperature and 438 the supernatant was decanted. Pelleted cells were re-suspended in 250µL PBS. Two 439 microliters of re-suspended cells were aliquoted on to 30 well microscope slides 440 (TEKDON incorporated, Slide ID:30-30), allowed to air dry and fixed in acetone for 441 10min at room temperature. Slides were washed in a 50mL conical tube containing 442 PBS for 5min at room temperature three times before primary antibody was applied 443 and incubated for 1-2 hours at 37°C. The slides were washed three times with PBS at 444 room temperature for 5 minutes. Anti-mouse or anti-rabbit secondary antibody was 445 applied where appropriate and incubated for 30 minutes at 37°C. Slides were rinsed 446 with PBS at room temperature for 5 minutes three times. The SlowFade™ Antifade Kit 447 was used as a mounting medium as well as to stain the cellular DNA with DAPI. Slides 448 were visualised on a Nikon Eclipse 80i microscope with Nikon Intensilight C-HGFI at 449 10X magnification and imaged with Nikon Digital Sight camera using NIS Elements 450 Imaging Software (v.3.22.15). Stem cell differentiation. The spent medium of W1 iPSCs was removed and the cells 463 were treated with ACCUTASEä and incubated for 7min at 37°C, 20% O2, 5% CO2. 464 Cells were dislodged, transferred to a 15mL conical tube, spun at 250 x g for 5 min at 465 room temperature, the supernatant was decanted and pelleted cells were re-466 suspended in 3mL TeSRä-E8ä in the presence of 10µM Y-27632. Cells were later 467 seeded on 6 wells plates coated with 1% Geltrexä at a density of 2 x 10 5 cells/well in 468 TeSRä-E8ä supplemented with Y-27632 and kept at 37°C, 5% CO2. Two days later, 469 spent medium was removed and replaced with differentiation medium (DMEM/F12, 470 1% ITS, 0.001nM isoprenaline, 100ng/mL BMP-4, 20ng/mL bFGF and 0.1µM Retinoic 471 acid) and incubated at 37°C, 20% O2, 5% CO2 for four days. Cells were then imaged 472 and maintained in culture or the RNA was extracted for qPCR to assess iPSC markers Infection quantification. Infectious particle quantification was determined via plaque 498 assay. Briefly, BHK21 cells were seeded at 2 x 10 5 cells/well in a 24 well plate in RMPI 499 medium supplemented with 2% FCS and 1% penicillin/streptomycin. Once confluency 500 was reached, the BHK21 cells were infected with a 10-fold serial dilution of 100µL of 501 virus and incubated at 37°C, 20% O2, 5% CO2 for 1 hour, with plate agitation at 15 502 minute intervals. Subsequently, viral inoculum was removed, 0.8% carboxymethyl 503 cellulose (CMC) in RPMI medium supplemented with 3% FCS and 504 penicillin/streptomycin was added and plates were incubated at 37°C, 20% O2, 5% 505 CO2 for six days. Cells were then fixed in 20% formalin for at least 30 minutes before 506 rinsing with water. Plates treated with 1% crystal violet (Sigma-Aldrich), washed and 507 air-dried to count plaques. 508 Viral genome copy number was quantified by qRT-PCR using CDC primers and 510 probes as previously described for dengue virus (39) and zika virus (40). Briefly, 511 virus genomic RNA was extracted from the supernatant using QIAamp Viral RNA 512 Mini Kit according to manufacturer's instructions. Purified RNA was then quantified 513 by qPCR following the qScript One-Step RT-PCR Kit protocol using the CDC 514 specified primers and probes on the LightCycler ® 480 II. Quantification and statistical analysis -All statistical analyses were performed 530 using GraphPad Prism (v.8.2.1). Student t-test was used and p-value of ≤ 0.05 was 531 considered significant (ns > 0.05, *p £ 0.05, **p £ 0.01, ***p £ 0.001, ****p £ 0.0001).  MRC-5 derived iPSCs