Employing epigenetic memory and native instructive stimuli to stimulate iPS-NLC differentiation

Notochordal cells (NCs) are linked to a healthy intervertebral disc (IVD), and are considered a promising candidate for cell-based therapies. However, NCs are scarcely available as they are lost early in life, and attempts at in vitro expansion have failed because NCs lose their specific phenotype. The production of notochordal-like cells (NLCs) from human induced pluripotent stem cells (hiPSCs) is a viable alternative. Therefore, this study aimed to build on the tissue-specific epigenetic memory of hiPSCs derived from IVD-progenitor cells (TIE2+-cells) and the instructive capacity of decellularized notochordal cell-derived matrix (dNCM)2 to improve hiPSC differentiation towards mature, healthy matrix-producing NLCs. hiPSCs were generated from TIE2+-IVD cells of three adult donors. As a comparison donor-matched minimally invasive peripheral blood mononuclear (PBM)-derived iPSCs were used. Firstly, hiPSCs were differentiated into mesendodermal progenitors by Wnt pathway activation (N2B27 medium + 3µM CHIR99021)1 for 2 days. Thereafter, the cells were further driven towards the NC-lineage by transfection with synthetic NOTO mRNA1 and matured by switching to a 3D-cell pellet culture in discogenic medium containing 10ng/mL TGF-β1 or 3mg/mL dNCM until day 28. Read-outs included cell morphology, gene and protein expression and matrix deposition. Both TIE2+- and PBM-cell derived hiPSC showed successful differentiation towards mesendodermal progenitors 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 and Nodal signaling (LEF1, NODAL) and mesendodermal markers (FOXA2, TBXT) was detected, compared to mTeSR1 controls. This was confirmed by immuno-stains for FOXA2 and TBXT. On day 3, we detected a significant increase in NOTO mRNA levels in all donor lines after transfection compared to untransfected cell pellets. 3D-pellets of all donor lines showed glycosaminoglycan (GAG)- and collagen type II-rich areas after dNCM- but not TGF-β1-treatment on day 28. This was confirmed with the DMMB-assay, showing a significantly increased GAG content in the 3D-pellets treated with dNCM compared to TGF-β1. Next to that, TIE2+-cell derived iPSC pellets contained a significantly higher GAG content after dNCM-treatment compared to the PBM-cell derived hiPSC pellets. Immunohistochemical evaluation showed a heterogeneous cell population including cells positive for chondrogenic- (ACAN, SOX9), NPC/NC- (panKRT, T), and IVD progenitor- markers (CD24, TIE2). In conclusion, using tissue-specific TIE2+-cell derived hiPSCs combined with dNCM-treatment may allow for an improved differentiation capacity indicated by the increased deposition of GAG and collagen type II-rich matrix. However, the obtained cell population is still very heterogeneous and further transcriptome analysis could unravel whether the 3D-pellets contain cells which were successfully driven towards the notochordal-lineage and how these can be enriched based on unique NC-specific markers. Next to that, delineating which epigenetic features are retained after reprogramming of these two cell lines, could shed light on the observed differences in their differentiation capacity. These insights could be used for further optimization of iPS-NLC differentiation and allow for a more purified population of mature, healthy matrix-producing NLCs. This work was funded by Horizon 2020 (no. 825925) and the Dutch Arthritis Society (LLP22). References 1Colombier, P. et al. (2020). NOTO transcription factor directs human induced pluripotent stem cell-derived mesendoderm progenitors to a notochordal fate. Cells, 9(2), 509. 2Bach, Frances C., et al. "Biologic canine and human intervertebral disc repair by notochordal cell-derived matrix: from bench towards bedside." Oncotarget 9.41 (2018): 26507

The ACER pollen and charcoal database: a global resource to document vegetation and fire response to abrupt climate changes during the last glacial period

Quaternary records provide an opportunity to examine the nature of the vegetation and fire responses to rapid past climate changes comparable in velocity and magnitude to those expected in the 21st-century. The best documented examples of rapid climate change in the past are the warming events associated with the Dansgaard–Oeschger (D–O) cycles during the last glacial period, which were sufficiently large to have had a potential feedback through changes in albedo and greenhouse gas emissions on climate. Previous reconstructions of vegetation and fire changes during the D–O cycles used independently constructed age models, making it difficult to compare the changes between different sites and regions. Here, we present the ACER (Abrupt Climate Changes and Environmental Responses) global database, which includes 93 pollen records from the last glacial period (73–15 ka) with a temporal resolution better than 1000 years, 32 of which also provide charcoal records. A harmonized and consistent chronology based on radiometric dating (14C, 234U∕230Th, optically stimulated luminescence (OSL), 40Ar∕39Ar-dated tephra layers) has been constructed for 86 of these records, although in some cases additional information was derived using common control points based on event stratigraphy. The ACER database compiles metadata including geospatial and dating information, pollen and charcoal counts, and pollen percentages of the characteristic biomes and is archived in Microsoft AccessTM at https://doi.org/10.1594/PANGAEA.870867

Production of Embryonic and Fetal-Like Red Blood Cells from Human Induced Pluripotent Stem Cells

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications

Revealing the last 13,500 years of environmental history from the multiproxy record of a mountain lake (Lago Enol, northern Iberian Peninsula)

This is the author's accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s10933-009-9387-7.We present the Holocene sequence from Lago Enol (43°16′N, 4°59′W, 1,070 m a.s.l.), Cantabrian Mountains, northern Spain. A multiproxy analysis provided comprehensive information about regional humidity and temperature changes. The analysis included sedimentological descriptions, physical properties, organic carbon and carbonate content, mineralogy and geochemical composition together with biological proxies including diatom and ostracod assemblages. A detailed pollen study enabled reconstruction of variations in vegetation cover, which were interpreted in the context of climate changes and human impact. Four distinct stages were recognized for the last 13,500 years: (1) a cold and dry episode that includes the Younger Dryas event (13,500–11,600 cal. year BP); (2) a humid and warmer period characterizing the onset of the Holocene (11,600–8,700 cal. year BP); (3) a tendency toward a drier climate during the middle Holocene (8,700–4,650 cal. year BP); and (4) a return to humid conditions following landscape modification by human activity (pastoral activities, deforestation) in the late Holocene (4,650–2,200 cal. year BP). Superimposed on relatively stable landscape conditions (e.g. maintenance of well established forests), the typical environmental variability of the southern European region is observed at this site.The Spanish Inter-Ministry Commission of Science and Technology (CICYT), the Spanish National Parks agency, the European Commission, the Spanish Ministry of Science, and the European Social Fund

The ACER pollen and charcoal database: a global resource to document vegetation and fire response to abrupt climate changes during the last glacial period

Quaternary records provide an opportunity to examine the nature of the vegetation and fire responses to rapid past climate changes comparable in velocity and magnitude to those expected in the 21st-century. The best documented examples of rapid climate change in the past are the warming events associated with the Dansgaard–Oeschger (D–O) cycles during the last glacial period, which were sufficiently large to have had a potential feedback through changes in albedo and greenhouse gas emissions on climate. Previous reconstructions of vegetation and fire changes during the D–O cycles used independently constructed age models, making it difficult to compare the changes between different sites and regions. Here, we present the ACER (Abrupt Climate Changes and Environmental Responses) global database, which includes 93 pollen records from the last glacial period (73–15 ka) with a temporal resolution better than 1000 years, 32 of which also provide charcoal records. A harmonized and consistent chronology based on radiometric dating (14C, 234U∕230Th, optically stimulated luminescence (OSL), 40Ar∕39Ar-dated tephra layers) has been constructed for 86 of these records, although in some cases additional information was derived using common control points based on event stratigraphy. The ACER database compiles metadata including geospatial and dating information, pollen and charcoal counts, and pollen percentages of the characteristic biomes and is archived in Microsoft AccessTM at https://doi.org/10.1594/PANGAEA.870867

Revealing the last 13,500 years of environmental history from the multiproxy record of a mountain lake (Lago Enol, northern Iberian Peninsula)

This is the author's accepted manuscript. The final publication is available at Springer via http://dx.doi.org/10.1007/s10933-009-9387-7.We present the Holocene sequence from Lago Enol (43°16′N, 4°59′W, 1,070 m a.s.l.), Cantabrian Mountains, northern Spain. A multiproxy analysis provided comprehensive information about regional humidity and temperature changes. The analysis included sedimentological descriptions, physical properties, organic carbon and carbonate content, mineralogy and geochemical composition together with biological proxies including diatom and ostracod assemblages. A detailed pollen study enabled reconstruction of variations in vegetation cover, which were interpreted in the context of climate changes and human impact. Four distinct stages were recognized for the last 13,500 years: (1) a cold and dry episode that includes the Younger Dryas event (13,500–11,600 cal. year BP); (2) a humid and warmer period characterizing the onset of the Holocene (11,600–8,700 cal. year BP); (3) a tendency toward a drier climate during the middle Holocene (8,700–4,650 cal. year BP); and (4) a return to humid conditions following landscape modification by human activity (pastoral activities, deforestation) in the late Holocene (4,650–2,200 cal. year BP). Superimposed on relatively stable landscape conditions (e.g. maintenance of well established forests), the typical environmental variability of the southern European region is observed at this site.The Spanish Inter-Ministry Commission of Science and Technology (CICYT), the Spanish National Parks agency, the European Commission, the Spanish Ministry of Science, and the European Social Fund

The ACER pollen and charcoal database: A global resource to document vegetation and fire response to abrupt climate changes during the last glacial period

This is the final version of the article. Available from Copernicus Publications via the DOI in this record.Quaternary records provide an opportunity to examine the nature of the vegetation and fire responses to rapid past climate changes comparable in velocity and magnitude to those expected in the 21st-century. The best documented examples of rapid climate change in the past are the warming events associated with the Dansgaard-Oeschger (D-O) cycles during the last glacial period, which were sufficiently large to have had a potential feedback through changes in albedo and greenhouse gas emissions on climate. Previous reconstructions of vegetation and fire changes during the D-O cycles used independently constructed age models, making it difficult to compare the changes between different sites and regions. Here, we present the ACER (Abrupt Climate Changes and Environmental Responses) global database, which includes 93 pollen records from the last glacial period (73-15ka) with a temporal resolution better than 1000years, 32 of which also provide charcoal records. A harmonized and consistent chronology based on radiometric dating (14C, 234U/230Th, optically stimulated luminescence (OSL), 40Ar/39Ar-dated tephra layers) has been constructed for 86 of these records, although in some cases additional information was derived using common control points based on event stratigraphy. The ACER database compiles metadata including geospatial and dating information, pollen and charcoal counts, and pollen percentages of the characteristic biomes and is archived in Microsoft Access™ at https://doi.org/10.1594/PANGAEA.870867.The members of the ACER project wish to thank the QUEST-DESIRE (UK and France) bilateral project, the INQUA International Focus Group ACER and the INTIMATE-COST action for funding a suite of workshops to compile the ACER pollen and charcoal database and the workshop on ACER chronology that allow setting the basis for harmonizing the chronologies. Josué M. Polanco-Martinez was funded by a Basque Government postdoctoral fellowship (POS_2015_1_0006) and Sandy P. Harrison by the ERC Advanced Grant GC2.0: unlocking the past for a clearer future

Comparative Analysis of DNA Replication Timing Reveals Conserved Large-Scale Chromosomal Architecture

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome

Replication Timing: A Fingerprint for Cell Identity and Pluripotency

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify “replication timing fingerprints” unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization