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When self-renewing pluripotent cells receive a differentiation signal, ongoing cell duplication needs to be coordinated with entry into a differentiation program. Accordingly, transcriptional activation of lineage specifier genes and cell differentiation is confined to the G1 phase of the cell cycle by unknown mechanisms. We found that Polycomb repressive complex 2 (PRC2) subunits are differentially recruited to lineage specifier gene promoters across cell cycle in mouse embryonic stem cells (mESCs). Jarid2 and the catalytic subunit Ezh2 are markedly accumulated at target promoters during S and G2 phases, while the transcriptionally activating subunits EPOP and EloB are enriched during G1 phase. Fluctuations in the recruitment of PRC2 subunits promote changes in RNA synthesis and RNA polymerase II binding that are compromised in Jarid2 −/− mESCs. Overall, we show that differential recruitment of PRC2 subunits across cell cycle enables the establishment of a chromatin state that facilitates the induction of cell differentiation in G1 phase., This study was supported by the Spanish Ministry of Economy and Competitiveness (SAF2013-40891-R and BFU2016-75233-P) and the Andalusian Regional Government (PC-0246-2017). D.L. is a Ramón y Cajal researcher of the Spanish Ministry of Economy and Competitiveness (RYC-2012-10019).

Mammals optimize their physiology to the light–dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used na¨ıve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, Bmal12/2 mESCs express higher levels of Nanog protein and altered expression of pluripotencyassociated signalling pathways. Importantly, Bmal12/2 mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development., Ramon y Cajal grant of the Spanish ministry of economy and competitiveness RYC2012-10019, Spanish ministry of economy and competitiveness BFU2016-75233-P, Andalusian regional government PC-0246-2017, Fundacion Progreso y Salud (FPS), Instituto de Salud Carlos III European Union (EU) CPII17/00032 PI17/01574, University of Granada

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling.

Bernard Soulier Syndrome (BSS) is a rare autosomal platelet disorder characterized by mutations in the von Willebrand factor platelet receptor complex GPIb-V-IX. In this work we have generated an induced pluripotent stem cell (BSS3-PBMC-iPS4F8) from peripheral blood mononuclear cells of a BSS patient with a p.Phe55Ser mutation in the GPIX gene. Characterization of BSS3-PBMC-iPS4F8 showed that these cells maintained the original mutation present in the BSS patient, expressed pluripotent stem cell markers and were able to differentiate into the three germline layers. This new iPSC line will contribute to better understand the biology of BSS disease.

We generated an induced pluripotent stem cell (iPSC) line from a Bernard–Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Studies with different animal models have shown that the Notch ligand DLL4 has a key role in the development of the embryonic vasculature. Here we describe the generation and characterization of a human embryonic stem cell line and an induced pluripotent stem cell line that constitutively express short hairpin RNAs targeting DLL4 mRNA. These cells present reduced DLL4 expression at both protein and mRNA level, as well as a reduced induction of DLL4 target genes. They represent an ideal tool to study the role of DLL4 in human embryonic vascular and hematopoietic development.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients, This work was supported by the Ramon y Cajal (RYC-2015-18382) to PJR founded by the Ministry of Economy and Competitiveness; the Instituto de Salud Carlos III-FEDER (CP12/03175 and CPII17/00032) to V.R-M., (PI17/01311) to M.L.L and J.R., (PI17/01966; Fundación Mutua Madrileña AP172142019; Premio Lopez Borrasca SETH 2019; GRS2061/A/19) to J.M.B. and (CPII15/00018 and PI16/01340) to PJR; by the Chair "Doctors Galera-Requena in cancer stem cell research" (CMC-CTS963) to J.A.M. and C.G-L.

Bernard Soulier Syndrome (BSS) is an inherited rare platelet disorder characterized by mutations in the platelet glycoprotein complex GPIb-IX-V. We generated an induced pluripotent stem cell (iPSC) line from a BSS patient with a mutation p.Asn45Ser in the GPIX locus (BSS2-PBMC-iPS4F24). Peripheral blood mononuclear cells were reprogrammed using non-integrative viral transduction. Characterization of BSS2-PBMC-iPS4F24 included mutational analysis of GPIX locus, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vitro and in vivo differentiation studies. This iPSC line will provide a powerful tool to study the biology of BSS disease.