Differentiation of human induced pluripotent stem cells towards notochordal-like cells: the role of tissue source

INTRODUCTION: Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and they are considered an exciting target for cell-based therapy. However, NCs are scarcely available as they are lost early in life, and attempts at in vivoexpansion have failed because NCs lose their specific phenotype. The production of Notochordal-like cells (NLCs) from human induced pluripotent stem cells (iPSCs) is a viable alternative. However, current attempts have been challenged by the low differentiation efficiency into the NC lineage. Therefore, the aim of this study was to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD progenitor cells (TIE2+-cells) to improve hiPSC differentiation towards mature, matrix-producing NLCs. METHODS: hiPSCs were generated from TIE2⁺ cells of three adult donors. As a comparison, donormatched minimally invasive peripheral blood mononuclear (PBM) cell-derived iPSCs were used. Firstly, the iPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)¹. Thereafter, the cells were further driven towards the NClineage by transfection with synthetic NOTO mRNA¹ and further matured using a 3D pellet culture in discogenic medium containing 10ng/mL TGF-β1. Read-out parameters included cell morphology, gene and protein expression and matrix deposition. RESULTS: Both TIE2⁺ and PBM cell-derived hiPSC showed successful differentiation towards mesendodermal progenitor cells following Wnt activation on day 2, indicated by the cells moving out of the colonies after CHIR stimulation. Accordingly, a decreased gene expression of pluripotency markers (OCT4, SOX2, NANOG), and upregulation of Wnt-target genes (LEF1, NODAL) and mesendodermal markers (TBXT, FOXA2, TBX6) was observed compared to mTESR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. At day 3, we confirmed a 9-fold increase in NOTO mRNA levels after transfection in all donor lines. At day 28, the appearance of vacuolated NLCs was observed in both TIE2⁺ and PBM cell-derived pellet cultures confirming successful commitment towards the NC-lineage. Interestingly, while DMMB-assay detected GAG deposition in both lines, a significant increase in GAG content was seen in the TIE2⁺ cell-derived pellets. DISCUSSION & CONCLUSIONS: Tissue-specific TIE2⁺ cell-derived iPSCs may allow for an improved iPSNLC differentiation efficiency, indicated by the increased potency for deposition of GAG-rich matrix. Detailed analysis of the phenotypic markers and matrix deposited at the end of the 28 day maturation is ongoing to further document the phenotype of these iPS-NLCs. Delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the differences in their differentiation capacity. REFERENCES: ¹Colombier et al., 202

Improvement of the assembly of heterozygous genomes of non-model organisms, a case study of the genomes of two Spodoptera frugiperda host strains

International audienceThe extraction of biological information from the draft genomes of non-model organisms may result in unattainable, incomplete, or even wrong conclusions. In particular, the combination of a high level of heterozygosity and short reads sequencing may have major impact in the annotation of genes [1,2]. This wrong gene content assessment is usually the consequence of the high fragmentation of the genome sequence but it may also come from an overestimation of the genome size. The latter because the assembly of an heterozygous region for which there is a significant divergence between the two haplotypes leads sometimes to the construction of two different contigs, instead of one consensus sequence. To date, new assemblers such as Platanus [3], have been developed in regard to heterozygous data. But, the complete re-assembly of a genome leading to new automatic and manual annotations process is very cost-effective, and may still produce erroneous scaffolds and annotations. Thus, we set up a « soft » method to detect and correct false duplications due to heterozygosity in draft assemblies. In addition, to the identification and removal of the allelic regions (i.e. unmerged haplotypes), our protocol is able to relocate and merge supernumerary gene annotations.We applied this method as a pre-requisite for the comparison of the genomes of 2 Spodoptera frugiperda (Lepidoptera: Noctuidae) strains, in the frame of the WGS project supported by the Fall armyworm International Public Consortium (FAW-IPC). This moth is a well-known pest of crops throughout the Western hemisphere. This species consists of two strains adapted to different larval host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. While, the paired-end reads of the rice-variant have been directly assembled using Platanus [3], we cleaned up and corrected the first release of the corn-variant, leading to a drastic reduction of the genome assembly, with the removal of 88Mbp (17%) and the increase of the N50 from 39,593 to 52,781bp. The suppressed fragments included 3,746 gene predictions; about 80% of them have been either relocated or merged with their complementary allele. Subsequently, in order to identify new candidate genes or genomic regions involved in the host-plant adaptation, we compared the genomes and proteomes of the 2 different strains in order to identify orthologous genes, collinear regions and genome rearrangements, taking into consideration the inflated occurrence of splitted genes due to the high fragmentation of the genome

Spodoptera frugiperda (Lepidoptera: Noctuidae) host-plant variants: two host strains or two distinct species?

International audienceThe moth Spodoptera frugiperda is a well-known pest of crops throughout the Americas, which consists of two strains adapted to different host-plants: the first feeds preferentially on corn, cotton and sorghum whereas the second is more associated with rice and several pasture grasses. Though morphologically indistinguishable, they exhibit differences in their mating behavior, pheromone compositions, and show development variability according to the host-plant. Though the latter suggest that both strains are different species, this issue is still highly controversial because hybrids naturally occur in the wild, not to mention the discrepancies among published results concerning mating success between the two strains. In order to clarify the status of the two host-plant strains of S. frugiperda, we analyze features that possibly reflect the level of post-zygotic isolation: (1) first generation (F1) hybrid lethality and sterility; (2) patterns of meiotic segregation of hybrids in reciprocal second generation (F2), as compared to the meiosis of the two parental strains. We found a significant reduction of mating success in F1 in one direction of the cross and a high level of microsatellite markers showing transmission ratio distortion in the F2 progeny. Our results support the existence of post-zygotic reproductive isolation between the two laboratory strains and are in accordance with the marked level of genetic differentiation that was recovered between individuals of the two strains collected from the field. Altogether these results provide additional evidence in favor of a sibling species status for the two strains

Unconventional phenomenology of a minimal two-Higgs-doublet model

Two-Higgs-doublet models (2HDM) are simple extensions of the Standard Model (SM) where the scalar sector is enlarged by adding a weak doublet. As a result, the Higgs potential depends in general on several free parameters which have to be carefully chosen to give predictions consistent with the current precision data. We consider a 2HDM invariant under a twisted custodial symmetry and depending only on three extra parameters beyond the SM ones. This model naturally features an inverted mass spectrum with a light pseudoscalar state and a heavy SM-like Higgs boson. We thoroughly analyze direct and indirect constraints and present a few unconventional though promising signatures at the LHC.Comment: 37 pages, 20 figure

Sex-related differences in aging rate are associated with sex chromosome system in amphibians

Sex-related differences in mortality are widespread in the animal kingdom. Although studies have shown that sex determination systems might drive lifespan evolution, sex chromosome influence on aging rates have not been investigated so far, likely due to an apparent lack of demographic data from clades including both XY (with heterogametic males) and ZW (heterogametic females) systems. Taking advantage of a unique collection of capture-recapture datasets in amphibians, a vertebrate group where XY and ZW systems have repeatedly evolved over the past 200 million years, we examined whether sex heterogamy can predict sex differences in aging rates and lifespans. We showed that the strength and direction of sex differences in aging rates (and not lifespan) differ between XY and ZW systems. Sex-specific variation in aging rates was moderate within each system, but aging rates tended to be consistently higher in the heterogametic sex. This led to small but detectable effects of sex chromosome system on sex differences in aging rates in our models. Although preliminary, our results suggest that exposed recessive deleterious mutations on the X/Z chromosome (the "unguarded X/Z effect") or repeat-rich Y/W chromosome (the "toxic Y/W effect") could accelerate aging in the heterogametic sex in some vertebrate clades.Peer reviewe

Meeting Report: Aging Research and Drug Discovery

Aging is the single largest risk factor for most chronic diseases, and thus possesses large socioeconomic interest to continuously aging societies. Consequently, the field of aging research is expanding alongside a growing focus from the industry and investors in aging research. This year's 8th Annual Aging Research and Drug Discovery ARDD) meeting was organized as a hybrid meeting from August 30th to September 3rd 2021 with more than 130 attendees participating on-site at the Ceremonial Hall at University of Copenhagen, Denmark, and 1800 engaging online. The conference comprised of presentations from 75 speakers focusing on new research in topics including mechanisms of aging and how these can be modulated as well as the use of AI and new standards of practices within aging research. This year, a longevity workshop was included to build stronger connections with the clinical community