formation-human-thymus-organoids-three-dimensional-fibrin

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064

Nantes Université, CHU Nantes, INSERM, CNRS, BioCore, SFR Bonamy

Despite advances in the field of somatic cell reprogramming, an understanding and exploration of the underlying mechanisms governing this process are only recently emerging. It is now increasingly apparent that key sequential events correlate with the reprogramming process; a process previously thought to be random and unpredictable is now looking, to a greater extent, defined and controlled. Herein, we will review the key cellular and molecular events associated with the reprogramming process, giving an integrative and conciliatory view of the different studies addressing the mechanism of nuclear reprogramming.

Phases of reprogramming.

While somatic cell reprogramming is now part of our text books, we ignore most of the mechanisms governing this cellular transformation. The most enigmatic question is why only rare cells undergo reprogramming, and whether this is governed by stochastc or deterministic events. In the late 2012, several major studies have addressed this question through a clonal analysis of the reprogramming process in murine MEF. In this mini-review, we describe these results and discuss future perspectives based on these date to optimize and secure the derivation of iPSC.

[Reprogramming equals gambling?].

Two studies by Sakurai et al. (2014) and Hu et al. (2014) in this issue of Cell Stem Cell add a new level of understanding to the mesenchymal-to-epithelial transition taking place during reprogramming, showing how this morphological transformation is promoted by Tet enzymes and blocked by kinase-dependent cytoskeletal organization.

How Tets and cytoskeleton dynamics MET in reprogramming.

Molecular Mechanisms of Stem Cell Pluripotency and Cell Fate Specification.

Induced pluripotent stem cells (iPSCs) have considerably impacted human developmental biology and regenerative medicine, notably because they circumvent the use of cells of embryonic origin and offer the potential to generate patient-specific pluripotent stem cells. However, conventional reprogramming protocols produce developmentally advanced, or primed, human iPSCs (hiPSCs), restricting their use to post-implantation human development modeling. Hence, there is a need for hiPSCs resembling preimplantation naive epiblast. Here, we develop a method to generate naive hiPSCs directly from somatic cells, using OKMS overexpression and specific culture conditions, further enabling parallel generation of their isogenic primed counterparts. We benchmark naive hiPSCs against human preimplantation epiblast and reveal remarkable concordance in their transcriptome, dependency on mitochondrial respiration and X-chromosome status. Collectively, our results are essential for the understanding of pluripotency regulation throughout preimplantation development and generate new opportunities for disease modeling and regenerative medicine.

Parallel derivation of isogenic human primed and naive induced pluripotent stem cells.

Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.

Urine-derived cells provide a readily accessible cell type for feeder-free mRNA reprogramming.

Human trophoblast stem cells (hTSCs) derived from blastocysts and first-trimester cytotrophoblasts offer an unprecedented opportunity to study the placenta. However, access to human embryos and first-trimester placentas is limited, thus preventing the establishment of hTSCs from diverse genetic backgrounds associated with placental disorders. Here, we show that hTSCs can be generated from numerous genetic backgrounds using post-natal cells via two alternative methods: (1) somatic cell reprogramming of adult fibroblasts with OCT4, SOX2, KLF4, MYC (OSKM) and (2) cell fate conversion of naive and extended pluripotent stem cells. The resulting induced/converted hTSCs recapitulated hallmarks of hTSCs including long-term self-renewal, expression of specific transcription factors, transcriptomic signature, and the potential to differentiate into syncytiotrophoblast and extravillous trophoblast cells. We also clarified the developmental stage of hTSCs and show that these cells resemble day 8 cytotrophoblasts. Altogether, hTSC lines of diverse genetic origins open the possibility to model both placental development and diseases in a dish.

Induction of Human Trophoblast Stem Cells from Somatic Cells and Pluripotent Stem Cells.

Understanding lineage specification during human pre-implantation development is a gateway to improving assisted reproductive technologies and stem cell research. Here we employ pseudotime analysis of single-cell RNA sequencing (scRNA-seq) data to reconstruct early mouse and human embryo development. Using time-lapse imaging of annotated embryos, we provide an integrated, ordered, and continuous analysis of transcriptomics changes throughout human development. We reveal that human trophectoderm/inner cell mass transcriptomes diverge at the transition from the B2 to the B3 blastocyst stage, just before blastocyst expansion. We explore the dynamics of the fate markers IFI16 and GATA4 and show that they gradually become mutually exclusive upon establishment of epiblast and primitive endoderm fates, respectively. We also provide evidence that NR2F2 marks trophectoderm maturation, initiating from the polar side, and subsequently spreads to all cells after implantation. Our study pinpoints the precise timing of lineage specification events in the human embryo and identifies transcriptomics hallmarks and cell fate markers.

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification.

Cell reprogramming has allowed unprecedented access to human development, from virtually any genome. However, reprogramming yields pluripotent stem cells that can differentiate into all cells that form a fetus, but not extraembryonic annexes. Therefore, a cellular model allowing study of placental development from a broad genomic repertoire is lacking. Here, we describe an optimized protocol to reprogram somatic cells into human induced trophoblast stem cells (hiTSCs) and convert pluripotent stem cells into human converted TSCs (hcTSCs). This protocol enables much-needed genome-specific placental disease modeling. We also detail extravillous trophoblast and syncytiotrophoblast differentiation protocols from hiTSCs and hcTSCs, a necessary step to validate these cells. In total, this protocol takes 4 months and requires advanced cell culture skills, comparable to those necessary for somatic cell reprogramming into human induced pluripotent stem cells.

Induction of human trophoblast stem cells.

Trophoblasts are specialized epithelial cells that perform critical functions during blastocyst implantation and mediate maternal-fetal communication during pregnancy. However, our understanding of human trophoblast biology remains limited since access to first-trimester placental tissue is scarce, especially between the first and fourth weeks of development. Moreover, animal models inadequately recapitulate unique aspects of human placental physiology. In the mouse system, the isolation of self-renewing trophoblast stem cells has provided a valuable in vitro model system of placental development, but the derivation of analogous human trophoblast stem cells (hTSCs) has remained elusive until recently. Building on a landmark study reporting the isolation of bona fide hTSCs from blastocysts and first-trimester placental tissues in 2018, several groups have developed methods to derive hTSCs from pluripotent and somatic cell sources. Here we review the biological and molecular properties that define authentic hTSCs, the trophoblast potential of distinct pluripotent states, and methods for inducing hTSCs in somatic cells by direct reprogramming. The generation of hTSCs from pluripotent and somatic cells presents exciting opportunities to elucidate the molecular mechanisms of human placental development and the etiology of pregnancy-related diseases.

Accessing the human trophoblast stem cell state from pluripotent and somatic cells.

Studying human embryo development, most particularly around the time of implantation, is a challenge, yet it is necessary to improve assisted reproduction techniques. In this issue, Yu et al. and Karvas et al. improve integrated stem cell models, called blastoids, to model the peri-implantation human embryo.

Having a blast(oid): Modeling human embryo peri-implantation development with blastoids.

Despite over 40 years following the first birth from medically assisted reproduction (MAR) technologies, mechanisms underlying the key developmental events during the first 7 days of human development, such as signaling pathway contribution, are remaining a mystery. An in-depth mechanistic understanding of how the human preimplantation embryo develops would support the optimization of embryo quality assessment methods and culturing conditions, thereby increasing the success rate of MAR. However, the limited availability of human embryos, legitimate ethical concerns, and regulations still present an obstacle toward our advancement of knowledge. Stem cell-based embryonic models, including blastoids than model blastocysts, offer unprecedented opportunities to fill knowledge gaps and complement animal models. Blastoids' predictive power depends on how faithfully they recapitulate the blastocyst. Here, we review the state of the art of human pre- and peri-implantation development and outline the specificities of human embryo research to clarify the framework for blastoid research.

An update on human pre- and peri-implantation development: a blueprint for blastoids.

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.

Laurent David

Nantes Université, CHU Nantes, INSERM, Center for Research in Transplantation and Translational Immunology, UMR 1064

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels