@article {70333, title = {Midgestation metabolic constraint in purine metabolism drives distinct strategies for placenta and fetal growth}, journal = {bioRxiv}, year = {2026}, month = {2026 Mar 18}, abstract = {

Purine nucleotides are essential for mammalian development1,2. Purine monophosphates support cell signaling and proliferation and are synthesized by cells through either de novo synthesis or a salvage pathway3. We previously identified a midgestational metabolic transition in mice (gestational days gd10.5{\textendash}11.5) characterized by changes in purine metabolism4. Midgestation is a period of rapid growth for placenta and embryo, yet it remains unclear how the placental tissues expand without directly competing with the embryo for biosynthetic resources. Here, we show that this midgestational metabolic transition is associated with a marked reduction in embryonic expression of purine salvage enzymes, which constrains embryonic metabolism and leads to different strategies for purine synthesis between the placenta and embryo. Midgestation embryos are unable to engage the purine salvage pathway even when de novo purine synthesis is blocked either in vivo or in ex utero embryo culture, whereas placental tissue and trophoblasts retain the capacity to use either pathway. Disruption of de novo purine synthesis in mice causes reduced embryonic growth, impaired axial elongation, and abnormal brain and placental development, which are only partially rescued by supplementation with purine salvage precursors. In human placenta, trophoblast stem cells readily switch between the de novo and salvage pathways based on nutrient availability, and syncytiotrophoblasts (STB) preferentially rely on the salvage pathway. We identified guanosine monophosphate (GMP) as a metabolic checkpoint regulating STB differentiation, with insufficient GMP levels causing degradation of the small GTPase Rheb and failure of mTOR activation. Supplementation of purine salvage substrates restored GMP synthesis and STB differentiation in humans, but not mice. Further, in vivo measurements in humans revealed that maternal circulating hypoxanthine decreases during pregnancy and is further reduced in women with clinically small placentas, highlighting the role of hypoxanthine in supporting placental growth. These results uncover compartmentalized purine salvage between the embryo and placenta as a mechanism that limits competition for biosynthetic resources and enables coordinated growth during mammalian development.

}, doi = {10.64898/2026.03.18.712680}, url = {https://www.biorxiv.org/content/early/2026/03/18/2026.03.18.712680}, author = {Xu, Weizhi and De La Cruz, Nancy and Woods, Andrea and Lokshtanov, Dmitry and Gao, Shihong and Khan, Nawal and Wright, Sylvia and Florian-Rodriguez, Maria E. and McIntire, Donald D. and Duryea, Elaine L and Nelson, David B. and Spong, Catherine Y. and Herrera, Christina L. and Hanna, Jacob H. and Srivatsan, Sanjay and Aguilera-Castrejon, Alejandro and Solmonson, Ashley} } @article {70339, title = {Reconstituting mouse embryogenesis Ex utero from Gastrulation to fetal development reveals maternally independent metabolic programs}, journal = {bioRxiv}, year = {2026}, month = {2026 Mar 17}, abstract = {

Mammalian development takes place inside the maternal uterus, creating technological constraints that make difficult the study of embryogenesis in live developing embryos. A central challenge for understanding the role of metabolism in mammalian development is discriminating placental and uterine-regulated signals from embryo-intrinsic processes independent of maternal influence, a process that until now has remained inseparable during gastrulation and organogenesis1{\textendash}3. Ex utero culture systems allowing continuous growth of embryos during pre-gastrulation to organogenesis4,5 offer a promising solution to this challenge. Here, we present optimized ex utero culture platforms that support faithful development of mouse embryos from gastrulation (embryonic day 6.5/7.5) through the fetal period (embryonic day \~12.5) and harnessed these platforms for dissecting metabolic transitions in vivo during embryogenesis independently of uterus and placenta. We characterized the metabolome of in utero and ex utero whole embryos, fetal organs and culture medium between embryonic days E6.5 and E12.5 by liquid chromatography mass-spectrometry (LC-MS) metabolomics, isotope tracing, and single cell transcriptomics. These datasets present a comprehensive overview of the dynamic embryonic metabolism during gastrulation and organogenesis in utero and ex utero. This analysis revealed that the midgestational metabolic switch occurring at E10.5-E11.5 is faithfully recapitulated ex utero, indicating that this transition is intrinsically programmed in embryonic tissues and does not require direct maternal or placental cues. Notably, oxygen availability modulated the extent of this transition, but elevated oxygen was insufficient to induce it prematurely, demonstrating that the switch is developmentally timed and only partially environmental-responsive. We further harnessed the ex utero platform for identifying and perturbing a mitochondrial redox shift at E7.5-E8.5 that is critical for developmental progress after gastrulation. These findings uncover the remarkable metabolic plasticity of the mammalian embryo, demonstrating its capacity to sustain growth independently of maternal inputs from the establishment of the body plan through the onset of the fetal period. Moreover, they highlight the use of long-term ex utero culture as a unique framework for dissecting the mechanisms that shape embryogenesis under physiological and experimentally perturbed conditions, while functionally uncoupling embryonic programs from maternal and placental influences.

}, doi = {10.64898/2026.03.17.710314}, url = {https://www.biorxiv.org/content/early/2026/03/18/2026.03.17.710314}, author = {Lokshtanov, Dmitry and Gao, Shihong Max and Xu, Weizhi and Kosman, Avraham and Roncato, Francesco and De La Cruz, Nancy and Khan, Nawal A. and Woods, Andrea and Campbell, Ian and Woehler, Andrew and Christoforou, Christina and Ding, Lang and Hu, Amy and Copeland, Monique and Wang, Lihua and Yang, Xiushuai and Raley, Castle and Delventhal, Kym M. and Herrera, Antonio and Valente, Alessandro and Wright, Sylvia and Gomez-Cesar, Elidet and Shlomo, Raanan and Golenchenko, Sergey and Oldak, Bernardo and Yilmaz, Alperen and Gurhan-Sebinc, Gulben and Comar, Mehmet-Yunus and Viukov, Sergey and Novershtern, Noa and Zhang, Hua and Duong, Thao and Li, Lingjun and Khatib, Nizar and Kakun, Reli Rachel and Espinosa-Medina, Isabel and Florian-Rodriguez, Maria E. and LaManno, Gioele and Tillberg, Paul W. and Wang, Meng C. and Maza, Itay and Srivatsan, Sanjay and Solmonson, Ashley and Hanna, Jacob H. and Aguilera-Castrejon, Alejandro} } @article {70053, title = {Transgene-free generation of mouse post-gastrulation whole embryo models solely from naive ESCs and iPSCs.}, journal = {Cell Stem Cell}, year = {2025}, month = {2025 Aug 06}, abstract = {

The generation of post-gastrulation stem cell-derived mouse embryo models (SEMs) exclusively from naive embryonic stem cells (nESCs) has underscored their ability to give rise to embryonic and extra-embryonic lineages. However, existing protocols for mouse SEMs rely on the separate induction of extra-embryonic lineages and on ectopic expression of transcription factors to induce nESC differentiation into trophectoderm (TE) or primitive endoderm (PrE). Here, we demonstrate that mouse nESCs and naive induced pluripotent stem cells (niPSCs) can be simultaneously co-induced, via signaling pathway modulation, to generate PrE and TE extra-embryonic cells that self-organize into embryonic day (E) 8.5-E8.75 transgene-free (TF) SEMs. We also devised an alternative condition (AC) naive media that in vitro stabilizes TF-SEM-competent OCT4+/NANOG+ nESC colonies that co-express antagonistic CDX2 and/or GATA6 extra-embryonic fate master regulators and self-renew while remaining poised for TE and PrE differentiation, respectively. These findings improve mouse SEM strategies and shed light on amplifying an inherent and dormant extra-embryonic plasticity of mouse naive pluripotent cells in vitro.

}, issn = {1875-9777}, doi = {10.1016/j.stem.2025.07.005}, url = {https://doi.org/10.1016/j.stem.2025.07.005}, author = {Yilmaz, Alperen and Gurhan, Gulben and Comar, Mehmet-Yunus and Viukov, Sergey and Serfaty, Inbal and Gayretli, Mert and Golenchenko, Sergey and Lokshtanov, Dmitry and Ashouokhi, Shahd and Polanco, Angel and Berlad, Idan and Ha, Tae-Won and Aguilera-Castrejon, Alejandro and Tarazi, Shadi and Cohen, Marina and Livnat, Nir and Kumar, Komal and Cholakkal, Hisham and Levy, Nathan and Yosef, Nir and Khatib, Nizar and Kakun, Reli Rachel and Kedmi, Merav and Nachman, Inbal Bolocan and Keren-Shaul, Hadas and Addadi, Yoseph and Orenbuch, Ayelet-Hashahar and Korovin, Karina and Molchadsky, Alina and Hochedlinger, Konrad and Gafni, Ohad and Maza, Itay and Novershtern, Noa and Oldak, Bernardo and Hanna, Jacob H} } @article {69698, title = {Temporal BMP4 effects on mouse embryonic and extraembryonic developmentAbstract}, journal = {Nature}, volume = {634}, year = {2024}, month = {09-2024}, pages = {652 - 661}, abstract = {

The developing placenta, which in mice originates through the extraembryonic ectoderm (ExE), is essential for mammalian embryonic development. Yet unbiased characterization of the differentiation dynamics\ of the ExE and its interactions with the embryo proper remains incomplete. Here we develop a temporal single-cell model of mouse gastrulation that maps continuous and parallel differentiation in embryonic and extraembryonic lineages. This is matched with a three-way perturbation approach to target signalling from the embryo proper, the ExE alone, or both. We show that ExE specification involves early spatial and transcriptional bifurcation of uncommitted ectoplacental cone cells and chorion progenitors. Early BMP4 signalling from chorion progenitors is required for proper differentiation of uncommitted ectoplacental cone cells and later for their specification towards trophoblast giant cells. We also find biphasic regulation by BMP4 in the embryo. The early ExE-originating BMP4 signal is necessary for proper mesoendoderm bifurcation and for\ allantois and primordial germ cell specification. However, commencing at embryonic day 7.5, embryo-derived BMP4 restricts the primordial germ cell pool size by favouring differentiation of their extraembryonic mesoderm precursors towards an allantois fate. ExE and embryonic tissues are therefore entangled in time, space and signalling axes, highlighting the importance of their integrated understanding and modelling in vivo and in vitro.

}, issn = {0028-0836}, doi = {10.1038/s41586-024-07937-5}, url = {https://www.nature.com/articles/s41586-024-07937-5https://www.nature.com/articles/s41586-024-07937-5.pdfhttps://www.nature.com/articles/s41586-024-07937-5.pdfhttps://www.nature.com/articles/s41586-024-07937-5}, author = {Hadas, Ron and Rubinstein, Hernan and Mittnenzweig, Markus and Mayshar, Yoav and Ben-Yair, Raz and Cheng, Saifeng and Aguilera-Castrejon, Alejandro and Reines, Netta and Orenbuch, Ayelet-Hashahar and Lifshitz, Aviezer and Chen, Dong-Yuan and Elowitz, Michael B. and Zernicka-Goetz, Magdalena and Hanna, Jacob H. and Tanay, Amos and Stelzer, Yonatan} } @article {69018, title = {Complete human day 14 post-implantation embryo models from na{\"\i}ve ES cells}, journal = {Nature}, year = {2023}, month = {09/2023}, abstract = {

The ability to study human post-implantation development remains limited due to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (i.e., embryonic disk, bilaminar disk, yolk- and chorionic sacs, surrounding trophoblasts) remain lacking2. Mouse na{\"\i}ve embryonic stem cells (ESCs) have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation mouse Structured Stem cell-based Embryo Models with spatially organized morphogenesis (SEMs)3. Here, we extend these findings to humans, while using only genetically unmodified human na{\"\i}ve ESCs (in HENSM conditions)4. Such human fully integrated SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos including epiblast, hypoblast, extra-embryonic mesoderm, and trophoblast surrounding the latter layers. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days post-fertilization (dpf) (Carnegie stage 6a). This includes embryonic disk and bilaminar disk formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, PGC specification, polarized yolk sac with visceral and parietal endoderm, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, a trophoblast surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform may enable the experimental interrogation of previously inaccessible windows of human early post-implantation up to peri-gastrulation development.

}, issn = {0028-0836}, doi = {10.1038/s41586-023-06604-5}, url = {https://www.nature.com/articles/s41586-023-06604-5https://www.nature.com/articles/s41586-023-06604-5.pdfhttps://www.nature.com/articles/s41586-023-06604-5.pdfhttps://www.nature.com/articles/s41586-023-06604-5}, author = {Oldak, Bernardo and Wildschutz, Emilie and Bondarenko, Vladyslav and Comar, Mehmet-Yunus and Zhao, Cheng and Aguilera-Castrejon, Alejandro and Tarazi, Shadi and Viukov, Sergey and Pham, Thi Xuan Ai and Ashouokhi, Shahd and Lokshtanov, Dmitry and Roncato, Francesco and Ariel, Eitan and Rose, Max and Livnat, Nir and Shani, Tom and Joubran, Carine and Cohen, Roni and Addadi, Yoseph and Chemla, Muriel and Kedmi, Merav and Keren-Shaul, Hadas and Pasque, Vincent and Petropoulos, Sophie and Lanner, Fredrik and Novershtern, Noa and Hanna, Jacob H.} } @article {68984, title = {Embryo~model completes gastrulation to neurulation and organogenesis.}, journal = {Nature}, volume = {610}, year = {2022}, month = {10/2022}, pages = {143-153}, abstract = {

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

}, keywords = {Animals, Cell Lineage, Embryo, Mammalian, Embryonic Stem Cells, Endoderm, Gastrulation, Heart, Mesencephalon, Mice, Models, Biological, Neural Tube, Neurulation, Organogenesis, PAX6 Transcription Factor, Prosencephalon, Somites}, issn = {1476-4687}, doi = {10.1038/s41586-022-05246-3}, author = {Amadei, Gianluca and Handford, Charlotte E and Qiu, Chengxiang and De Jonghe, Joachim and Greenfeld, Hannah and Tran, Martin and Martin, Beth K and Chen, Dong-Yuan and Aguilera-Castrejon, Alejandro and Hanna, Jacob H and Elowitz, Michael B and Hollfelder, Florian and Shendure, Jay and Glover, David M and Zernicka-Goetz, Magdalena} } @article {68985, title = {Human primed and na{\"\i}ve PSCs are both able to differentiate into trophoblast stem cells.}, journal = {Stem Cell Reports}, volume = {17}, year = {2022}, month = {11/2022}, pages = {2484-2500}, abstract = {

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human na{\"\i}ve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor β (TGF-β) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human na{\"\i}ve PSCs expanded under human enhanced na{\"\i}ve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in\ vitro conversion into extra-embryonic trophoblast lineages.

}, keywords = {Blastocyst, Cell Differentiation, Female, Humans, Placenta, Pluripotent Stem Cells, Pregnancy, Trophoblasts}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2022.09.008}, author = {Viukov, Sergey and Shani, Tom and Bayerl, Jonathan and Aguilera-Castrejon, Alejandro and Oldak, Bernardo and Sheban, Daoud and Tarazi, Shadi and Stelzer, Yonatan and Hanna, Jacob H and Novershtern, Noa} } @article {68988, title = {Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis.}, journal = {J Vis Exp}, year = {2021}, month = {10/2021}, abstract = {

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

}, keywords = {Animals, Embryo Culture Techniques, Embryo, Mammalian, Embryonic Development, Female, Gastrulation, Mammals, Mice, Organogenesis, Pregnancy}, issn = {1940-087X}, doi = {10.3791/63160}, author = {Aguilera-Castrejon, Alejandro and Hanna, Jacob H} } @article {68989, title = {Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.}, journal = {Nature}, volume = {593}, year = {2021}, month = {05/2021}, pages = {119-124}, abstract = {

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis\ in mammals.

}, keywords = {Animals, Embryo Culture Techniques, Embryo, Mammalian, Embryonic Development, Female, Gastrulation, In Vitro Techniques, Male, Mice, Organogenesis, Time Factors, Uterus}, issn = {1476-4687}, doi = {10.1038/s41586-021-03416-3}, author = {Aguilera-Castrejon, Alejandro and Oldak, Bernardo and Shani, Tom and Ghanem, Nadir and Itzkovich, Chen and Slomovich, Sharon and Tarazi, Shadi and Bayerl, Jonathan and Chugaeva, Valeriya and Ayyash, Muneef and Ashouokhi, Shahd and Sheban, Daoud and Livnat, Nir and Lasman, Lior and Viukov, Sergey and Zerbib, Mirie and Addadi, Yoseph and Rais, Yoach and Cheng, Saifeng and Stelzer, Yonatan and Keren-Shaul, Hadas and Shlomo, Raanan and Massarwa, Rada and Novershtern, Noa and Maza, Itay and Hanna, Jacob H} } @article {68990, title = {Principles of signaling pathway modulation for enhancing human naive pluripotency induction.}, journal = {Cell Stem Cell}, volume = {28}, year = {2021}, month = {09/2021}, pages = {1549-1565.e12}, abstract = {

Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/β-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in\ vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.

}, keywords = {Animals, Cell Differentiation, Embryo, Mammalian, Humans, Mice, Pluripotent Stem Cells, Signal Transduction, Trophoblasts}, issn = {1875-9777}, doi = {10.1016/j.stem.2021.04.001}, author = {Bayerl, Jonathan and Ayyash, Muneef and Shani, Tom and Manor, Yair Shlomo and Gafni, Ohad and Massarwa, Rada and Kalma, Yael and Aguilera-Castrejon, Alejandro and Zerbib, Mirie and Amir, Hadar and Sheban, Daoud and Geula, Shay and Mor, Nofar and Weinberger, Leehee and Naveh Tassa, Segev and Krupalnik, Vladislav and Oldak, Bernardo and Livnat, Nir and Tarazi, Shadi and Tawil, Shadi and Wildschutz, Emilie and Ashouokhi, Shahd and Lasman, Lior and Rotter, Varda and Hanna, Suhair and Ben-Yosef, Dalit and Novershtern, Noa and Viukov, Sergey and Hanna, Jacob H} } @article {68991, title = {Context-dependent functional compensation between Ythdf mA reader proteins.}, journal = {Genes Dev}, volume = {34}, year = {2020}, month = {10/2020}, pages = {1373-1391}, abstract = {

The N6-methyladenosine (mA) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous mA-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers{\textquoteright} expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers{\textquoteright} role during early development that is gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.

}, keywords = {Animals, Cell Line, Dosage Compensation, Genetic, Embryonic Stem Cells, Fertility, Gametogenesis, Gene Deletion, Gene Expression Profiling, Gene Expression Regulation, Developmental, Methyltransferases, Mice, Mice, Knockout, RNA-Binding Proteins}, issn = {1549-5477}, doi = {10.1101/gad.340695.120}, author = {Lasman, Lior and Krupalnik, Vladislav and Viukov, Sergey and Mor, Nofar and Aguilera-Castrejon, Alejandro and Schneir, Dan and Bayerl, Jonathan and Mizrahi, Orel and Peles, Shani and Tawil, Shadi and Sathe, Shashank and Nachshon, Aharon and Shani, Tom and Zerbib, Mirie and Kilimnik, Itay and Aigner, Stefan and Shankar, Archana and Mueller, Jasmine R and Schwartz, Schraga and Stern-Ginossar, Noam and Yeo, Gene W and Geula, Shay and Novershtern, Noa and Hanna, Jacob H} } @article {68993, title = {Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules.}, journal = {Cell Stem Cell}, volume = {24}, year = {2019}, month = {02/2019}, pages = {328-341.e9}, abstract = {

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

}, keywords = {Animals, Cell Lineage, Cellular Reprogramming, Chromatin, Demethylation, Epigenesis, Genetic, Humans, Induced Pluripotent Stem Cells, Kruppel-Like Factor 4, Mice, Protein Binding, Proto-Oncogene Proteins c-myc, RNA, Transfer, Transcription Factors, Transcription, Genetic}, issn = {1875-9777}, doi = {10.1016/j.stem.2018.11.014}, author = {Zviran, Asaf and Mor, Nofar and Rais, Yoach and Gingold, Hila and Peles, Shani and Chomsky, Elad and Viukov, Sergey and Buenrostro, Jason D and Scognamiglio, Roberta and Weinberger, Leehee and Manor, Yair S and Krupalnik, Vladislav and Zerbib, Mirie and Hezroni, Hadas and Jaitin, Diego Adhemar and Larastiaso, David and Gilad, Shlomit and Benjamin, Sima and Gafni, Ohad and Mousa, Awni and Ayyash, Muneef and Sheban, Daoud and Bayerl, Jonathan and Aguilera-Castrejon, Alejandro and Massarwa, Rada and Maza, Itay and Hanna, Suhair and Stelzer, Yonatan and Ulitsky, Igor and Greenleaf, William J and Tanay, Amos and Trumpp, Andreas and Amit, Ido and Pilpel, Yitzhak and Novershtern, Noa and Hanna, Jacob H} } @article {68992, title = {Neuronal Transdifferentiation Potential of Human Mesenchymal Stem Cells from Neonatal and Adult Sources by a Small Molecule Cocktail.}, journal = {Stem Cells Int}, volume = {2019}, year = {2019}, month = {04/2019}, pages = {7627148}, abstract = {

Human mesenchymal stem cells (MSCs) are good candidates for brain cell replacement strategies and have already been used as adjuvant treatments in neurological disorders. MSCs can be obtained from many different sources, and the present study compares the potential of neuronal transdifferentiation in MSCs from adult and neonatal sources (Wharton{\textquoteright}s jelly (WhJ), dental pulp (DP), periodontal ligament (PDL), gingival tissue (GT), dermis (SK), placenta (PLAC), and umbilical cord blood (UCB)) with a protocol previously tested in bone marrow- (BM-) MSCs consisting of a cocktail of six small molecules: I-BET151, CHIR99021, forskolin, RepSox, Y-27632, and dbcAMP (ICFRYA). Neuronal morphology and the presence of cells positive for neuronal markers (TUJ1 and MAP2) were considered attributes of neuronal induction. The ICFRYA cocktail did not induce neuronal features in WhJ-MSCs, and these features were only partial in the MSCs from dental tissues, SK-MSCs, and PLAC-MSCs. The best response was found in UCB-MSCs, which was comparable to the response of BM-MSCs. The addition of neurotrophic factors to the ICFRYA cocktail significantly increased the number of cells with complex neuron-like morphology and increased the number of cells positive for mature neuronal markers in BM- and UCB-MSCs. The neuronal cells generated from UCB-MSCs and BM-MSCs showed increased reactivity of the neuronal genes TUJ1, MAP2, NF-H, NCAM, ND1, TAU, ENO2, GABA, and NeuN as well as down- and upregulation of MSC and neuronal genes, respectively. The present study showed marked differences between the MSCs from different sources in response to the transdifferentiation protocol used here. These results may contribute to identifying the best source of MSCs for potential cell replacement therapies.

}, issn = {1687-966X}, doi = {10.1155/2019/7627148}, author = {Cort{\'e}s-Medina, Lorena V and Pasantes-Morales, Herminia and Aguilera-Castrejon, Alejandro and Picones, Arturo and Lara-Figueroa, Cesar O and Luis, Enoch and Montesinos, Juan Jose and Cort{\'e}s-Morales, Victor A and De la Rosa Ruiz, M P and Hern{\'a}ndez-Est{\'e}vez, Erika and Bonifaz, Laura C and Alvarez-Perez, Marco Antonio and Ramos-Mandujano, Gerardo} } @article {68994, title = {Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency.}, journal = {Cell Stem Cell}, volume = {23}, year = {2018}, month = {09/2018}, pages = {412-425.e10}, abstract = {

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100\% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8\ days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

}, keywords = {Animals, Cells, Cultured, DNA Helicases, DNA-Binding Proteins, Female, GATA Transcription Factors, Induced Pluripotent Stem Cells, Male, Mi-2 Nucleosome Remodeling and Deacetylase Complex, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Knockout, Mice, Transgenic, Transcription Factors}, issn = {1875-9777}, doi = {10.1016/j.stem.2018.07.004}, author = {Mor, Nofar and Rais, Yoach and Sheban, Daoud and Peles, Shani and Aguilera-Castrejon, Alejandro and Zviran, Asaf and Elinger, Dalia and Viukov, Sergey and Geula, Shay and Krupalnik, Vladislav and Zerbib, Mirie and Chomsky, Elad and Lasman, Lior and Shani, Tom and Bayerl, Jonathan and Gafni, Ohad and Hanna, Suhair and Buenrostro, Jason D and Hagai, Tzachi and Masika, Hagit and Vainorius, Gintautas and Bergman, Yehudit and Greenleaf, William J and Esteban, Miguel A and Elling, Ulrich and Levin, Yishai and Massarwa, Rada and Merbl, Yifat and Novershtern, Noa and Hanna, Jacob H} } @article {68995, title = {Improved Proliferative Capacity of NP-Like Cells Derived from Human Mesenchymal Stromal Cells and Neuronal Transdifferentiation by Small Molecules.}, journal = {Neurochem Res}, volume = {42}, year = {2017}, month = {02/2017}, pages = {415-427}, abstract = {

Neural progenitors (NP), found in fetal and adult brain, differentiate into neurons potentially able to be used in cell replacement therapies. This approach however, raises technical and ethical problems which limit their potential therapeutic use. Alternately, NPs can be obtained by transdifferentiation of non-neural somatic cells evading these difficulties. Human bone marrow mesenchymal stromal cells (MSCs) are suggested to transdifferentiate into NP-like cells, which however, have a low proliferation capacity. The present study demonstrates the requisite of cell adhesion for proliferation and survival of NP-like cells and re-evaluates some neuronal features after differentiation by standard procedures. Mature neuronal markers, though, were not detected by these procedures. A chemical differentiation approach was used in this study to convert MSCs-derived NP-like cells into neurons by using a cocktail of six molecules, CHIR99021, I-BET151, RepSox, DbcAMP, forskolin and Y-27632, defined after screening combinations of 22 small molecules. Direct transdifferentiation of MSCs into neuronal cells was obtained with the small molecule cocktail, without requiring the NP-like intermediate stage.

}, keywords = {Adolescent, Adult, Amides, Cell Proliferation, Cell Transdifferentiation, Cells, Cultured, Colforsin, Drug Combinations, Heterocyclic Compounds, 4 or More Rings, Humans, Male, Mesenchymal Stem Cells, Neural Stem Cells, Neurons, Pyridines, Young Adult}, issn = {1573-6903}, doi = {10.1007/s11064-016-2086-7}, author = {Aguilera-Castrejon, Alejandro and Pasantes-Morales, Herminia and Montesinos, Juan Jose and Cort{\'e}s-Medina, Lorena V and Castro-Manrreza, Marta E and Mayani, H{\'e}ctor and Ramos-Mandujano, Gerardo} }