80 results on '"Lanner F"'
Search Results
2. Process Development and Manufacturing: RELEASE TESTING OF GMP MANUFACTURED CELLTHRPE1 FOR TREATMENT OF DRY AGE-RELATED MACULAR DEGENERATION
- Author
-
Vidal, L. Baque, primary, Main, H., additional, Reilly, H., additional, Hedenskog, M., additional, Beri, N., additional, Metzger, H., additional, Saietz, S., additional, Berggren, S., additional, Holm, A., additional, Eistrand, U., additional, Wrona, A., additional, von Halling Laier, C., additional, Efstathopoulos, P., additional, Jaberi, E., additional, Willenbrock, H., additional, Shimizu, Y., additional, Galler, G., additional, Elefante, F., additional, Jensen, L.B., additional, Nielsen, K., additional, Kragh, M., additional, Roubroeks, J., additional, Toft, D.B., additional, Villaescusa, J., additional, Christiansen, M.W., additional, André, H., additional, Kvanta, A., additional, Markland, K., additional, Blomberg, P., additional, and Lanner, F., additional
- Published
- 2023
- Full Text
- View/download PDF
3. O-096 The role of amnion and a human specific signaling centre in preimplantation development
- Author
-
Lanner, F, primary
- Published
- 2022
- Full Text
- View/download PDF
4. 56 Mammalian pre-implantation embryos at the single cell level: The bovine as a model for early human embryonic development
- Author
-
Ortega, N., primary, Ahola, V., additional, Plaza-Reyes, A., additional, Schell, J., additional, Kumar, P., additional, Jouneau, A., additional, Duranthon, V., additional, and Lanner, F., additional
- Published
- 2020
- Full Text
- View/download PDF
5. Complement system proteins in human embryonic stem cell-derived retinal pigment epithelial cells co-cultured with or without porcine retina
- Author
-
Mohlin, C., Petrus-Reurer, S., Lanner, F., Sandholm, K., Kvanta, A., Nilsson, Bo, Nilsson Ekdahl, Kristina, Mohlin, C., Petrus-Reurer, S., Lanner, F., Sandholm, K., Kvanta, A., Nilsson, Bo, and Nilsson Ekdahl, Kristina
- Published
- 2017
- Full Text
- View/download PDF
6. Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo.
- Author
-
Posfai, E, Petropoulos, S, de Barros, FRO, Schell, JP, Jurisica, I, Sandberg, R, Lanner, F, Rossant, J, Posfai, E, Petropoulos, S, de Barros, FRO, Schell, JP, Jurisica, I, Sandberg, R, Lanner, F, and Rossant, J
- Abstract
The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling.
- Published
- 2017
7. Notch Signaling Regulates Platelet-Derived Growth Factor Receptor-?² Expression in Vascular Smooth Muscle Cells
- Author
-
Jin, S., Hansson, E. M., Tikka, S., Lanner, F., Sahlgren, C., Farnebo, F., Baumann, M., Kalimo, H., and Lendahl, U.
- Published
- 2008
- Full Text
- View/download PDF
8. Complement system proteins in human embryonic stem cell-derived retinal pigment epithelial cells co-cultured with or without porcine retina
- Author
-
Mohlin, C., primary, Petrus-Reurer, S., additional, Lanner, F., additional, Sandholm, K., additional, Kvanta, A., additional, Nilsson, B., additional, and Ekdahl, K.N., additional
- Published
- 2017
- Full Text
- View/download PDF
9. Single-cell RNA sequencing: revealing human pre-implantation development, pluripotency and germline development
- Author
-
Petropoulos, S., primary, Panula, S. P., additional, Schell, J. P., additional, and Lanner, F., additional
- Published
- 2016
- Full Text
- View/download PDF
10. OR31-03 Single-Cell Profiling of Adult Human Ovarian Cortex Reveals Six Main Cell Types but No Germline Stem Cells
- Author
-
Wagner M, Yoshihara M, Douagi I, Damdimopoulos A, Panula S, Petropoulos S, Lu H, Pettersson K, Palm K, Katayama S, Hovatta O, Juha Kere, Lanner F, and Damdimopoulou P
11. Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States
- Author
-
Collier, AJ, Panula, SP, Schell, JP, Chovanec, P, Plaza Reyes, A, Petropoulos, S, Corcoran, AE, Walker, R, Douagi, I, Lanner, F, and Rugg-Gunn, PJ
- Subjects
cell surface markers ,blastocyst ,reprogramming ,antibody library ,differentiation ,embryonic stem cells ,pluripotency ,3. Good health - Abstract
Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.
12. Sex-biased gene expression during neural differentiation of human embryonic stem cells.
- Author
-
Pottmeier P, Nikolantonaki D, Lanner F, Peuckert C, and Jazin E
- Abstract
Sex differences in the developing human brain are primarily attributed to hormonal influence. Recently however, genetic differences and their impact on the developing nervous system have attracted increased attention. To understand genetically driven sexual dimorphisms in neurodevelopment, we investigated genome-wide gene expression in an in vitro differentiation model of male and female human embryonic stem cell lines (hESC), independent of the effects of human sex hormones. Four male and four female-derived hESC lines were differentiated into a population of mixed neurons over 37 days. Differential gene expression and gene set enrichment analyses were conducted on bulk RNA sequencing data. While similar differentiation tendencies in all cell lines demonstrated the robustness and reproducibility of our differentiation protocol, we found sex-biased gene expression already in undifferentiated ESCs at day 0, but most profoundly after 37 days of differentiation. Male and female cell lines exhibited sex-biased expression of genes involved in neurodevelopment, suggesting that sex influences the differentiation trajectory. Interestingly, the highest contribution to sex differences was found to arise from the male transcriptome, involving both Y chromosome and autosomal genes. We propose 13 sex-biased candidate genes (10 upregulated in male cell lines and 3 in female lines) that are likely to affect neuronal development. Additionally, we confirmed gene dosage compensation of X/Y homologs escaping X chromosome inactivation through their Y homologs and identified a significant overexpression of the Y-linked demethylase UTY and KDM5D in male hESC during neuron development, confirming previous results in neural stem cells. Our results suggest that genetic sex differences affect neuronal differentiation trajectories, which could ultimately contribute to sex biases during human brain development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pottmeier, Nikolantonaki, Lanner, Peuckert and Jazin.)
- Published
- 2024
- Full Text
- View/download PDF
13. Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells.
- Author
-
Baqué-Vidal L, Main H, Petrus-Reurer S, Lederer AR, Beri NE, Bär F, Metzger H, Zhao C, Efstathopoulos P, Saietz S, Wrona A, Jaberi E, Willenbrock H, Reilly H, Hedenskog M, Moussaud-Lamodière E, Kvanta A, Villaescusa JC, La Manno G, and Lanner F
- Subjects
- Humans, Aged, Cell Differentiation, Cryopreservation, Epithelial Cells, Retinal Pigments, Pluripotent Stem Cells, Macular Degeneration therapy
- Abstract
Background Aims: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease., Methods: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location., Results: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology., Conclusions: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
14. Complete human day 14 post-implantation embryo models from naive ES cells.
- Author
-
Oldak B, Wildschutz E, Bondarenko V, Comar MY, Zhao C, Aguilera-Castrejon A, Tarazi S, Viukov S, Pham TXA, Ashouokhi S, Lokshtanov D, Roncato F, Ariel E, Rose M, Livnat N, Shani T, Joubran C, Cohen R, Addadi Y, Chemla M, Kedmi M, Keren-Shaul H, Pasque V, Petropoulos S, Lanner F, Novershtern N, and Hanna JH
- Subjects
- Humans, Fertilization, Gastrulation, Germ Layers cytology, Germ Layers embryology, Trophoblasts cytology, Yolk Sac cytology, Yolk Sac embryology, Giant Cells cytology, Embryo Implantation, Embryo, Mammalian cytology, Embryo, Mammalian embryology, Embryonic Development, Human Embryonic Stem Cells cytology
- Abstract
The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation
1 . Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2 . Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3 . Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4 . Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development., (© 2023. The Author(s).)- Published
- 2023
- Full Text
- View/download PDF
15. Abnormal developmental trajectory and vulnerability to cardiac arrhythmias in tetralogy of Fallot with DiGeorge syndrome.
- Author
-
Chan CH, Lam YY, Wong N, Geng L, Zhang J, Ahola V, Zare A, Li RA, Lanner F, Keung W, and Cheung YF
- Subjects
- Humans, Arrhythmias, Cardiac etiology, Myocytes, Cardiac, DiGeorge Syndrome complications, DiGeorge Syndrome genetics, Tetralogy of Fallot complications, Induced Pluripotent Stem Cells, RGS Proteins
- Abstract
Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Ventricular dysfunction and cardiac arrhythmias are well-documented complications in patients with repaired TOF. Whether intrinsic abnormalities exist in TOF cardiomyocytes is unknown. We establish human induced pluripotent stem cells (hiPSCs) from TOF patients with and without DiGeorge (DG) syndrome, the latter being the most commonly associated syndromal association of TOF. TOF-DG hiPSC-derived cardiomyocytes (hiPSC-CMs) show impaired ventricular specification, downregulated cardiac gene expression and upregulated neural gene expression. Transcriptomic profiling of the in vitro cardiac progenitors reveals early bifurcation, as marked by ectopic RGS13 expression, in the trajectory of TOF-DG-hiPSC cardiac differentiation. Functional assessments further reveal increased arrhythmogenicity in TOF-DG-hiPSC-CMs. These findings are found only in the TOF-DG but not TOF-with no DG (ND) patient-derived hiPSC-CMs and cardiac progenitors (CPs), which have implications on the worse clinical outcomes of TOF-DG patients., (© 2023. Springer Nature Limited.)
- Published
- 2023
- Full Text
- View/download PDF
16. In Reply: Revisiting Claims of the Continued Absence of Functional Germline Stem Cells in Adult Ovaries.
- Author
-
Yoshihara M, Wagner M, Damdimopoulos A, Zhao C, Petropoulos S, Katayama S, Kere J, Lanner F, and Damdimopoulou P
- Subjects
- Female, Humans, Stem Cells, Ovary, Germ Cells
- Published
- 2023
- Full Text
- View/download PDF
17. The Continued Absence of Functional Germline Stem Cells in Adult Ovaries.
- Author
-
Yoshihara M, Wagner M, Damdimopoulos A, Zhao C, Petropoulos S, Katayama S, Kere J, Lanner F, and Damdimopoulou P
- Subjects
- Adult, Female, Humans, Middle Aged, Ovarian Follicle, Germ Cells, Stem Cells metabolism, Ovary, Oogenesis physiology
- Abstract
Ovaries are central to development, fertility, and reproduction of women. A particularly interesting feature of ovaries is their accelerated aging compared to other tissues, leading to loss of function far before other organs senesce. The limited pool of ovarian follicles is generated before birth and once exhausted, menopause will inevitably commence around the age of 50 years marking the end of fertility. Yet, there are reports suggesting the presence of germline stem cells and neo-oogenesis in adult human ovaries. These observations have fueled a long debate, created experimental fertility treatments, and opened business opportunities. Our recent analysis of cell types in the ovarian cortex of women of fertile age could not find evidence of germline stem cells. Like before, our work has been met with critique suggesting methodological shortcomings. We agree that excellence starts with methods and welcome discussion on the pros and cons of different protocols. In this commentary, we discuss the recent re-interpretation of our work., (© The Author(s) 2022. Published by Oxford University Press.)
- Published
- 2023
- Full Text
- View/download PDF
18. Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.
- Author
-
Koel M, Krjutškov K, Saare M, Samuel K, Lubenets D, Katayama S, Einarsdottir E, Vargas E, Sola-Leyva A, Lalitkumar PG, Gemzell-Danielsson K, Blesa D, Simon C, Lanner F, Kere J, Salumets A, and Altmäe S
- Abstract
Study Question: Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells?, Summary Answer: A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin β1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted., What Is Known Already: The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted., Study Design Size Duration: Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts., Participants/materials Setting Methods: Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion., Main Results and the Role of Chance: In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin β1 (ITGB1), basigin (BSG ) and osteopontin (SPP1) in the embryo implantation process were highlighted., Large Scale Data: RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929., Limitations Reasons for Caution: Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only., Wider Implications of the Findings: The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives., Study Funding/competing Interests: This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)
- Published
- 2022
- Full Text
- View/download PDF
19. Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation.
- Author
-
Petrus-Reurer S, Lederer AR, Baqué-Vidal L, Douagi I, Pannagel B, Khven I, Aronsson M, Bartuma H, Wagner M, Wrona A, Efstathopoulos P, Jaberi E, Willenbrock H, Shimizu Y, Villaescusa JC, André H, Sundstrӧm E, Bhaduri A, Kriegstein A, Kvanta A, La Manno G, and Lanner F
- Subjects
- Animals, Cell Differentiation genetics, Humans, Retinal Pigment Epithelium, Retinal Pigments, Human Embryonic Stem Cells, Macular Degeneration genetics, Macular Degeneration therapy
- Abstract
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during in vitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1
+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
20. Polycomb repressive complex 2 shields naïve human pluripotent cells from trophectoderm differentiation.
- Author
-
Kumar B, Navarro C, Winblad N, Schell JP, Zhao C, Weltner J, Baqué-Vidal L, Salazar Mantero A, Petropoulos S, Lanner F, and Elsässer SJ
- Subjects
- Cell Differentiation genetics, Embryonic Development, Histones genetics, Humans, Human Embryonic Stem Cells metabolism, Polycomb Repressive Complex 2 genetics, Polycomb Repressive Complex 2 metabolism
- Abstract
The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an 'activated' state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development., (© 2022. The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
21. A peek into the black box of human embryology.
- Author
-
Goedel A and Lanner F
- Published
- 2021
- Full Text
- View/download PDF
22. Refined transcriptional blueprint of human preimplantation embryos.
- Author
-
Weltner J and Lanner F
- Subjects
- Humans, Blastocyst, Embryonic Development
- Abstract
Despite being a biologically fundamental question, the precise timing of lineage specification during human preimplantation development remains elusive. In this issue of Cell Stem Cell, Meistermann et al. (2021) refine our view through time-lapse embryo staging and single-cell sequencing and challenge the concept of a human inner cell mass., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
23. Amnion signals are essential for mesoderm formation in primates.
- Author
-
Yang R, Goedel A, Kang Y, Si C, Chu C, Zheng Y, Chen Z, Gruber PJ, Xiao Y, Zhou C, Witman N, Eroglu E, Leung CY, Chen Y, Fu J, Ji W, Lanner F, Niu Y, and Chien KR
- Subjects
- Amnion embryology, Animals, Bone Morphogenetic Protein 4 metabolism, Embryonic Development, Female, Gene Expression Regulation, Developmental, LIM-Homeodomain Proteins genetics, LIM-Homeodomain Proteins metabolism, Macaca fascicularis genetics, Macaca fascicularis metabolism, Mesoderm metabolism, Pregnancy, Signal Transduction, Amnion metabolism, Macaca fascicularis embryology, Mesoderm embryology
- Abstract
Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
24. All models are wrong, but some are useful: Establishing standards for stem cell-based embryo models.
- Author
-
Posfai E, Lanner F, Mulas C, and Leitch HG
- Subjects
- Animals, Embryonic Development, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Epigenesis, Genetic, Humans, Reference Standards, Embryo, Mammalian physiology, Models, Biological
- Abstract
Detailed studies of the embryo allow an increasingly mechanistic understanding of development, which has proved of profound relevance to human disease. The last decade has seen in vitro cultured stem cell-based models of embryo development flourish, which provide an alternative to the embryo for accessible experimentation. However, the usefulness of any stem cell-based embryo model will be determined by how accurately it reflects in vivo embryonic development, and/or the extent to which it facilitates new discoveries. Stringent benchmarking of embryo models is thus an important consideration for this growing field. Here we provide an overview of means to evaluate both the properties of stem cells, the building blocks of most embryo models, as well as the usefulness of current and future in vitro embryo models., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
25. Evaluating totipotency using criteria of increasing stringency.
- Author
-
Posfai E, Schell JP, Janiszewski A, Rovic I, Murray A, Bradshaw B, Yamakawa T, Pardon T, El Bakkali M, Talon I, De Geest N, Kumar P, To SK, Petropoulos S, Jurisicova A, Pasque V, Lanner F, and Rossant J
- Subjects
- Animals, Blastomeres metabolism, Embryo, Mammalian metabolism, Embryonic Stem Cells metabolism, Female, Gene Expression Profiling, Gene Regulatory Networks, Male, Mice, Pluripotent Stem Cells metabolism, Single-Cell Analysis, Totipotent Stem Cells metabolism, Blastomeres cytology, Cell Differentiation, Cell Lineage genetics, Embryo, Mammalian cytology, Embryonic Stem Cells cytology, Pluripotent Stem Cells cytology, Totipotent Stem Cells cytology
- Abstract
Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.
- Published
- 2021
- Full Text
- View/download PDF
26. Preclinical safety studies of human embryonic stem cell-derived retinal pigment epithelial cells for the treatment of age-related macular degeneration.
- Author
-
Petrus-Reurer S, Kumar P, Padrell Sánchez S, Aronsson M, André H, Bartuma H, Plaza Reyes A, Nandrot EF, Kvanta A, and Lanner F
- Subjects
- Aged, Cell Differentiation, Human Embryonic Stem Cells cytology, Humans, Pluripotent Stem Cells cytology, Human Embryonic Stem Cells metabolism, Macular Degeneration therapy, Pluripotent Stem Cells metabolism
- Abstract
As pluripotent stem cell (PSC)-based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age-related macular degeneration is one of the first that has reached patients. We have previously established a xeno-free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)-derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole-genome sequencing (WGS) to assess genome stability, single-cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC-based therapies and the data support safety of the hESC-RPE cells generated through our in vitro differentiation methodology., (© 2020 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)
- Published
- 2020
- Full Text
- View/download PDF
27. Publisher Correction: Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells.
- Author
-
Reyes AP, Petrus-Reurer S, Sánchez SP, Kumar P, Douagi I, Bartuma H, Aronsson M, Westman S, Lardner E, André H, Falk A, Nandrot EF, Kvanta A, and Lanner F
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2020
- Full Text
- View/download PDF
28. Karolinska Institutet Human Embryonic Stem Cell Bank.
- Author
-
Main H, Hedenskog M, Acharya G, Hovatta O, and Lanner F
- Subjects
- Cell Culture Techniques, Cell Differentiation, Cell Line, Embryonic Stem Cells, Humans, Sweden, Human Embryonic Stem Cells
- Abstract
The Karolinska Institutet Human Embryonic Stem Cell Bank (KI Stem Cell Bank) was established at KI, Stockholm, Sweden, when the first human embryonic stem cell (hESC) line was derived by Professor Hovatta and colleagues in 2002. Since then, the bank has grown to include 60 hESC lines. From the very beginning the aim of the bank has been derivation of hESC lines suitable for clinical use. Step by step progress has been made towards this goal, including removal of xeno components, establishment of chemically defined conditions and Good Manufacturing Practice (GMP) compliancy. Today our bank includes such clinical grade hESC line, KARO1, derived and banked according to GMP guidelines. Many of the hESC lines in the bank have been distributed to the scientific community and are deposited in the Stockholm Medical Biobank available for research on collaborative basis., Competing Interests: Declaration of interests Authors declare no conflict of interest, (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
29. Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II.
- Author
-
Petrus-Reurer S, Winblad N, Kumar P, Gorchs L, Chrobok M, Wagner AK, Bartuma H, Lardner E, Aronsson M, Plaza Reyes Á, André H, Alici E, Kaipe H, Kvanta A, and Lanner F
- Subjects
- CRISPR-Associated Protein 9 metabolism, CRISPR-Cas Systems genetics, Cytotoxicity, Immunologic, Heterografts, Human Embryonic Stem Cells metabolism, Humans, Immunomodulation, Nuclear Proteins metabolism, Polymorphism, Single Nucleotide genetics, T-Lymphocytes metabolism, Trans-Activators metabolism, beta 2-Microglobulin metabolism, Epithelial Cells metabolism, Histocompatibility Antigens Class I metabolism, Histocompatibility Antigens Class II metabolism, Human Embryonic Stem Cells cytology, Retinal Pigment Epithelium cytology
- Abstract
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells could serve as a replacement therapy in advanced stages of age-related macular degeneration. However, allogenic hESC-RPE transplants trigger immune rejection, supporting a strategy to evade their immune recognition. We established single-knockout beta-2 microglobulin (SKO-B2M), class II major histocompatibility complex transactivator (SKO-CIITA) and double-knockout (DKO) hESC lines that were further differentiated into corresponding hESC-RPE lines lacking either surface human leukocyte antigen class I (HLA-I) or HLA-II, or both. Activation of CD4+ and CD8+ T-cells was markedly lower by hESC-RPE DKO cells, while natural killer cell cytotoxic response was not increased. After transplantation of SKO-B2M, SKO-CIITA, or DKO hESC-RPEs in a preclinical rabbit model, donor cell rejection was reduced and delayed. In conclusion, we have developed cell lines that lack both HLA-I and -II antigens, which evoke reduced T-cell responses in vitro together with reduced rejection in a large-eyed animal model., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
30. Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells.
- Author
-
Plaza Reyes A, Petrus-Reurer S, Padrell Sánchez S, Kumar P, Douagi I, Bartuma H, Aronsson M, Westman S, Lardner E, André H, Falk A, Nandrot EF, Kvanta A, and Lanner F
- Subjects
- Animals, CD56 Antigen, Embryonic Stem Cells, Humans, Laminin genetics, Macular Degeneration metabolism, Rabbits, Retinal Pigment Epithelium metabolism, Biomarkers metabolism, Cell Differentiation physiology, Epithelial Cells metabolism, Neurons metabolism, Retinal Pigments metabolism
- Abstract
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.
- Published
- 2020
- Full Text
- View/download PDF
31. Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation.
- Author
-
Fernandez-Alonso R, Bustos F, Budzyk M, Kumar P, Helbig AO, Hukelmann J, Lamond AI, Lanner F, Zhou H, Petsalaki E, and Findlay GM
- Subjects
- Animals, Cell Differentiation genetics, Embryonic Development genetics, Embryonic Development physiology, Ephrin-A2, Fibroblast Growth Factor 4 metabolism, Humans, Ligands, MAP Kinase Signaling System, Mice, Phosphorylation, Receptor, EphA2 genetics, Signal Transduction, Cell Differentiation physiology, Embryo, Mammalian metabolism, Embryonic Stem Cells metabolism, Proteomics methods, Receptor, EphA2 metabolism
- Abstract
Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.
- Published
- 2020
- Full Text
- View/download PDF
32. Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells.
- Author
-
Wagner M, Yoshihara M, Douagi I, Damdimopoulos A, Panula S, Petropoulos S, Lu H, Pettersson K, Palm K, Katayama S, Hovatta O, Kere J, Lanner F, and Damdimopoulou P
- Subjects
- Adult, Biomarkers metabolism, Cells, Cultured, DEAD-box RNA Helicases immunology, DEAD-box RNA Helicases metabolism, Female, Gene Expression Profiling, Humans, Sex Reassignment Procedures, Transcriptome, Oogonial Stem Cells, Ovary cytology, Single-Cell Analysis methods
- Abstract
The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.
- Published
- 2020
- Full Text
- View/download PDF
33. Human induced pluripotent stem cells from two azoospermic patients with Klinefelter syndrome show similar X chromosome inactivation behavior to female pluripotent stem cells.
- Author
-
Panula S, Kurek M, Kumar P, Albalushi H, Padrell Sánchez S, Damdimopoulou P, Olofsson JI, Hovatta O, Lanner F, and Stukenborg JB
- Subjects
- Adult, Cell Differentiation, Female, Fibroblasts metabolism, Genotype, Histones metabolism, Humans, Male, Phenotype, Sex Factors, Teratoma metabolism, Transcriptome, Azoospermia genetics, Chromosomes, Human, X, Klinefelter Syndrome genetics, Pluripotent Stem Cells cytology, X Chromosome Inactivation
- Abstract
Study Question: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?, Summary Answer: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features., What Is Known Already: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture., Study Design, Size, Duration: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor., Participants/materials, Setting, Methods: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives., Main Results and the Role of Chance: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS., Limitations, Reasons for Caution: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments., Wider Implications of the Findings: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis., Study Funding/competing Interest(s): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2019
- Full Text
- View/download PDF
34. iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture.
- Author
-
Gelali E, Girelli G, Matsumoto M, Wernersson E, Custodio J, Mota A, Schweitzer M, Ferenc K, Li X, Mirzazadeh R, Agostini F, Schell JP, Lanner F, Crosetto N, and Bienko M
- Subjects
- A549 Cells, Chromosome Mapping methods, Chromosomes, Human genetics, Fibroblasts, Human Embryonic Stem Cells, Humans, Oligonucleotides genetics, Real-Time Polymerase Chain Reaction methods, Research Design, DNA Probes genetics, Databases, Nucleic Acid, Genome, Human genetics, In Situ Hybridization, Fluorescence methods
- Abstract
DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.
- Published
- 2019
- Full Text
- View/download PDF
35. A speculative outlook on embryonic aneuploidy: Can molecular pathways be involved?
- Author
-
Tšuiko O, Jatsenko T, Parameswaran Grace LK, Kurg A, Vermeesch JR, Lanner F, Altmäe S, and Salumets A
- Subjects
- Animals, Female, Humans, Oocytes metabolism, Oocytes pathology, Pregnancy, Aneuploidy, Blastocyst metabolism, Blastocyst pathology, Embryonic Development, Genomic Instability, Pregnancy Complications genetics, Pregnancy Complications metabolism, Pregnancy Complications pathology
- Abstract
The journey of embryonic development starts at oocyte fertilization, which triggers a complex cascade of events and cellular pathways that guide early embryogenesis. Recent technological advances have greatly expanded our knowledge of cleavage-stage embryo development, which is characterized by an increased rate of whole-chromosome losses and gains, mixoploidy, and atypical cleavage morphokinetics. Embryonic aneuploidy significantly contributes to implantation failure, spontaneous miscarriage, stillbirth or congenital birth defects in both natural and assisted human reproduction. Essentially, early embryo development is strongly determined by maternal factors. Owing to considerable limitations associated with human oocyte and embryo research, the use of animal models is inevitable. However, cellular and molecular mechanisms driving the error-prone early stages of development are still poorly described. In this review, we describe known events that lead to aneuploidy in mammalian oocytes and preimplantation embryos. As the processes of oocyte and embryo development are rigorously regulated by multiple signal-transduction pathways, we explore the putative role of signaling pathways in genomic integrity maintenance. Based on the existing evidence from human and animal data, we investigate whether critical early developmental pathways, like Wnt, Hippo and MAPK, together with distinct DNA damage response and DNA repair pathways can be associated with embryo genomic instability, a question that has, so far, remained largely unexplored., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
36. Functional genetics of early human development.
- Author
-
Ortega NM, Winblad N, Plaza Reyes A, and Lanner F
- Subjects
- Embryo, Mammalian, Germ Cells growth & development, Humans, Zygote, Cell Differentiation genetics, Embryonic Development genetics, Genome, Human genetics, X Chromosome Inactivation genetics
- Abstract
Understanding the genetic underpinning of early human development is of great interest not only for basic developmental and stem cell biology but also for regenerative medicine, infertility treatments, and better understanding the causes of congenital disease. Our current knowledge has mainly been generated with the use of laboratory animals, especially the mouse. While human and mouse early development present morphological resemblance, we know that the timing of the events as well as the cellular and genetic mechanisms that control fundamental processes are distinct between the species. The rapid technological development of single-cell sequencing and genome editing together with novel stem cell models of the early human embryo has made it feasible and relevant to perform functional genetic studies directly in human cells and embryos. In this review we will discuss these latest advances where combined transcriptional analysis and genome engineering has begun to shed new insights into the key processes of zygotic genome activation, lineage specification, X-chromosome inactivation and postimplantation development including primordial germ cell specification in the human embryo., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
37. Time Matters: Gene Editing at the Mouse 2-Cell Embryo Stage Boosts Knockin Efficiency.
- Author
-
Plaza Reyes A and Lanner F
- Subjects
- Animals, Mice, Microinjections, CRISPR-Cas Systems, Gene Editing
- Abstract
Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
38. Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy.
- Author
-
Petrus-Reurer S, Bartuma H, Aronsson M, Westman S, Lanner F, and Kvanta A
- Subjects
- Animals, Cell Differentiation physiology, Disease Models, Animal, Geographic Atrophy pathology, Humans, Rabbits, Retina cytology, Retinal Pigment Epithelium cytology, Transplantation, Heterologous, Geographic Atrophy diagnosis, Human Embryonic Stem Cells transplantation, Macular Degeneration therapy, Retina transplantation, Retinal Pigment Epithelium transplantation
- Abstract
Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.
- Published
- 2018
- Full Text
- View/download PDF
39. The E-cadherin/AmotL2 complex organizes actin filaments required for epithelial hexagonal packing and blastocyst hatching.
- Author
-
Hildebrand S, Hultin S, Subramani A, Petropoulos S, Zhang Y, Cao X, Mpindi J, Kalloniemi O, Johansson S, Majumdar A, Lanner F, and Holmgren L
- Subjects
- Angiomotins, Animals, Blastocyst cytology, Carrier Proteins genetics, Cell Line, Epithelial Cells cytology, Epithelium metabolism, Gene Expression, Gene Knockout Techniques, Humans, Intercellular Junctions metabolism, Mice, Multiprotein Complexes metabolism, Protein Binding, Skin cytology, Skin metabolism, Stress, Mechanical, Zebrafish, Actin Cytoskeleton metabolism, Blastocyst metabolism, Cadherins metabolism, Carrier Proteins metabolism, Epithelial Cells metabolism
- Abstract
Epithelial cells connect via cell-cell junctions to form sheets of cells with separate cellular compartments. These cellular connections are essential for the generation of cellular forms and shapes consistent with organ function. Tissue modulation is dependent on the fine-tuning of mechanical forces that are transmitted in part through the actin connection to E-cadherin as well as other components in the adherens junctions. In this report we show that p100 amotL2 forms a complex with E-cadherin that associates with radial actin filaments connecting cells over multiple layers. Genetic inactivation or depletion of amotL2 in epithelial cells in vitro or zebrafish and mouse in vivo, resulted in the loss of contractile actin filaments and perturbed epithelial packing geometry. We further showed that AMOTL2 mRNA and protein was expressed in the trophectoderm of human and mouse blastocysts. Genetic inactivation of amotL2 did not affect cellular differentiation but blocked hatching of the blastocysts from the zona pellucida. These results were mimicked by treatment with the myosin II inhibitor blebbistatin. We propose that the tension generated by the E-cadherin/AmotL2/actin filaments plays a crucial role in developmental processes such as epithelial geometrical packing as well as generation of forces required for blastocyst hatching.
- Published
- 2017
- Full Text
- View/download PDF
40. Biotechnology: At the heart of gene edits in human embryos.
- Author
-
Winblad N and Lanner F
- Subjects
- Humans, Biotechnology, Embryo, Mammalian
- Published
- 2017
- Full Text
- View/download PDF
41. Comprehensive Cell Surface Protein Profiling Identifies Specific Markers of Human Naive and Primed Pluripotent States.
- Author
-
Collier AJ, Panula SP, Schell JP, Chovanec P, Plaza Reyes A, Petropoulos S, Corcoran AE, Walker R, Douagi I, Lanner F, and Rugg-Gunn PJ
- Subjects
- Cell Line, Humans, Pluripotent Stem Cells cytology, Antigens, Differentiation biosynthesis, Gene Expression Profiling, Gene Expression Regulation physiology, Membrane Proteins biosynthesis, Pluripotent Stem Cells metabolism
- Abstract
Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
42. Overexpression of Trophoblast Stem Cell-Enriched MicroRNAs Promotes Trophoblast Fate in Embryonic Stem Cells.
- Author
-
Nosi U, Lanner F, Huang T, and Cox B
- Subjects
- Animals, Cell Line, Cells, Cultured, Embryonic Stem Cells metabolism, Gene Expression Regulation, Developmental, Mice, MicroRNAs genetics, Trophoblasts metabolism, Cell Lineage, Embryonic Stem Cells cytology, MicroRNAs metabolism, Trophoblasts cytology
- Abstract
The first cell fate choice of the preimplantation embryo generates the extraembryonic trophoblast and embryonic epiblast lineages. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) can be utilized to investigate molecular mechanisms of this first cell fate decision. It has been established that ESCs can be induced to acquire trophoblast lineage characteristics upon manipulation of lineage-determining transcription factors. Here, we have interrogated the potential of microRNAs (miRNAs) to drive trans-differentiation of ESCs into the trophoblast lineage. Analysis of gene expression data identified a network of TSC-enriched miRNAs that were predicted to target mRNAs enriched in ESCs. Ectopic expression of these miRNAs in ESCs resulted in a stable trophoblast phenotype, supported by gene expression changes and in vivo contribution potential. This process is highly miRNA-specific and dependent on Hdac2 inhibition. Our experimental evidence suggests that these miRNAs promote a mural trophectoderm (TE)-like cell fate with physiological properties that differentiate them from the polar TE., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
43. Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo.
- Author
-
Posfai E, Petropoulos S, de Barros FRO, Schell JP, Jurisica I, Sandberg R, Lanner F, and Rossant J
- Subjects
- Animals, Gene Expression Profiling, Gene Expression Regulation, Developmental, Genes, Reporter, Green Fluorescent Proteins analysis, Green Fluorescent Proteins genetics, Hippo Signaling Pathway, Mice, Sequence Analysis, RNA, Ectoderm embryology, Protein Serine-Threonine Kinases metabolism, Signal Transduction
- Abstract
The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling.
- Published
- 2017
- Full Text
- View/download PDF
44. Integration of Subretinal Suspension Transplants of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells in a Large-Eyed Model of Geographic Atrophy.
- Author
-
Petrus-Reurer S, Bartuma H, Aronsson M, Westman S, Lanner F, André H, and Kvanta A
- Subjects
- Animals, Cell Culture Techniques, Disease Models, Animal, Humans, Injections, Intraocular, Rabbits, Epithelial Cells transplantation, Geographic Atrophy therapy, Human Embryonic Stem Cells cytology, Retinal Pigment Epithelium cytology
- Abstract
Purpose: Subretinal suspension transplants of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have the capacity to form functional monolayers in naive eyes. We explore hESC-RPE integration when transplanted in suspension to a large-eyed model of geographic atrophy (GA)., Methods: Derivation of hESC-RPE was performed in a xeno-free and defined manner. Subretinal bleb injection of PBS or sodium iodate (NaIO3) was used to induce a GA-like phenotype. Suspensions of hESC-RPE were transplanted to the subretinal space of naive or PBS-/NaIO3-treated rabbits using a transvitreal pars plana technique. Integration of hESC-RPE was monitored by multimodal real-time imaging and by immunohistochemistry., Results: Subretinal blebs of PBS or NaIO3 caused different degrees of outer neuroretinal degeneration, RPE hyperautofluorescence, focal RPE loss, and choroidal atrophy; that is, hallmark characteristics of GA. In nonpretreated naive eyes, hESC-RPE integrated as subretinal monolayers with preserved overlying photoreceptors, yet not in areas with outer neuroretinal degeneration and native RPE loss. When transplanted to eyes with PBS-/NaIO3-induced degeneration, hESC-RPE failed to integrate., Conclusions: In a large-eyed preclinical model, subretinal suspension transplants of hESC-RPE did not integrate in areas with GA-like degeneration.
- Published
- 2017
- Full Text
- View/download PDF
45. Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.
- Author
-
Plaza Reyes A and Lanner F
- Subjects
- Animals, Embryo, Mammalian, Humans, CRISPR-Cas Systems genetics, Embryo Research ethics, Embryonic Development genetics, Gene Editing ethics, Gene Editing statistics & numerical data, Gene Editing trends
- Abstract
Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof., (© 2017. Published by The Company of Biologists Ltd.)
- Published
- 2017
- Full Text
- View/download PDF
46. Single-cell sequencing of the small-RNA transcriptome.
- Author
-
Faridani OR, Abdullayev I, Hagemann-Jensen M, Schell JP, Lanner F, and Sandberg R
- Subjects
- Cell Line, Tumor, Cells, Cultured, Humans, High-Throughput Nucleotide Sequencing methods, Human Embryonic Stem Cells physiology, MicroRNAs genetics, Neoplasms, Experimental genetics, Sequence Analysis, RNA methods, Transcriptome genetics
- Abstract
Little is known about the heterogeneity of small-RNA expression as small-RNA profiling has so far required large numbers of cells. Here we present a single-cell method for small-RNA sequencing and apply it to naive and primed human embryonic stem cells and cancer cells. Analysis of microRNAs and fragments of tRNAs and small nucleolar RNAs (snoRNAs) reveals the potential of microRNAs as markers for different cell types and states.
- Published
- 2016
- Full Text
- View/download PDF
47. Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation.
- Author
-
Chen G, Schell JP, Benitez JA, Petropoulos S, Yilmaz M, Reinius B, Alekseenko Z, Shi L, Hedlund E, Lanner F, Sandberg R, and Deng Q
- Subjects
- Animals, Cells, Cultured, Embryonic Stem Cells metabolism, Female, Gene Expression Regulation, Developmental, Male, Mice, Mice, Inbred C57BL, Pluripotent Stem Cells metabolism, Single-Cell Analysis, Transcriptome, Cell Differentiation, Embryonic Stem Cells cytology, Pluripotent Stem Cells cytology, X Chromosome Inactivation
- Abstract
Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we "digitalized" XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI., (© 2016 Chen et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2016
- Full Text
- View/download PDF
48. The human PRD-like homeobox gene LEUTX has a central role in embryo genome activation.
- Author
-
Jouhilahti EM, Madissoon E, Vesterlund L, Töhönen V, Krjutškov K, Plaza Reyes A, Petropoulos S, Månsson R, Linnarsson S, Bürglin T, Lanner F, Hovatta O, Katayama S, and Kere J
- Subjects
- Animals, Cell Line, Electrophoretic Mobility Shift Assay, Fluorescent Antibody Technique, Indirect, Gene Expression Regulation, Developmental, Homeodomain Proteins genetics, Humans, Mice, Polymerase Chain Reaction, Protein Isoforms genetics, Blastocyst metabolism, Embryonic Stem Cells metabolism, Homeodomain Proteins metabolism, Protein Isoforms metabolism
- Abstract
Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
49. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.
- Author
-
Petropoulos S, Edsgärd D, Reinius B, Deng Q, Panula SP, Codeluppi S, Reyes AP, Linnarsson S, Sandberg R, and Lanner F
- Published
- 2016
- Full Text
- View/download PDF
50. Corrigendum: Characterization and target genes of nine human PRD-like homeobox domain genes expressed exclusively in early embryos.
- Author
-
Madissoon E, Jouhilahti EM, Vesterlund L, Töhönen V, Krjutškov K, Petropoulos S, Einarsdottir E, Linnarsson S, Lanner F, Månsson R, Hovatta O, Bürglin TR, Katayama S, and Kere J
- Published
- 2016
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.