Molécules du CMH, bactéries et auto-immunité (contributions à l'étude des arthrites associées à HLA-B27)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Engineering of midbrain organoids containing long-lived dopaminergic neurons

The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease

Generation of human induced pluripotent stem cell line UNIGEi003-A from skin fibroblasts of an apparently healthy male donor

Dermal fibroblasts isolated from an apparently healthy 50-year-old man were successfully transformed into induced pluripotent stem cells (iPSCs) by using the integration-free CytoTune-iPS Sendai Reprogramming method. The generated iPSC line has been expanded under feeder-free conditions and displayed all hallmarks of a standard pluripotent stem cell line such as a normal karyotype, expression of pluripotent factors and differentiation capacity into the three germ layers

Interplay of TRIM28 and DNA methylation in controlling human endogenous retroelements

Reverse transcription-derived sequences account for at least half of the human genome. Although these retroelements are formidable motors of evolution, they can occasionally cause disease, and accordingly are inactivated during early embryogenesis through epigenetic mechanisms. In the mouse, at least for endogenous retroviruses, important mediators of this process are the tetrapod-specific KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor TRIM28. The present study demonstrates that KRAB/TRIM28-mediated regulation is responsible for controlling a very broad range of human-specific endogenous retroelements (EREs) in human embryonic stem (ES) cells and that it exerts, as a consequence, a marked effect on the transcriptional dynamics of these cells. It further reveals reciprocal dependence between TRIM28 recruitment at specific families of EREs and DNA methylation. It finally points to the importance of persistent TRIM28-mediated control of ERE transcriptional impact beyond their presumed inactivation by DNA methylation

Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed. Here, we propose a suicide gene approach, based on cell cycle-dependent promoter Ki67-driven expression of herpes simplex virus thymidine kinase (HSV-TK). We generated a PSC line expressing this construct and induced neural differentiation. In vitro, proliferating PSC and early neural precursor cells (NPC) were killed by exposure to ganciclovir. In vivo, transplantation of PSC led to tumor formation, which was prevented by early ganciclovir treatment. Transplanted NPC did not lead to tumor formation and their survival and neural maturation were not affected by ganciclovir. In conclusion, the cell cycle promoter-driven suicide gene approach described in this study allows killing of proliferating undifferentiated precursor cells without expression of the suicide gene in mature neurons. This approach could also be of use for other stem cell-based therapies where the final target consists of postmitotic cells

Human neural organoids for studying brain cancer and neurodegenerative diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing

Cardiac neural crest cells: their rhombomeric specification, migration, and association with heart and great vessel anomalies

Outflow tract abnormalities are the most frequent congenital heart defects. These are due to the absence or dysfunction of the two main cell types, i.e., neural crest cells and secondary heart field cells that migrate in opposite directions at the same stage of development. These cells directly govern aortic arch patterning and development, ascending aorta dilatation, semi-valvular and coronary artery development, aortopulmonary septation abnormalities, persistence of the ductus arteriosus, trunk and proximal pulmonary arteries, sub-valvular conal ventricular septal/rotational defects, and non-compaction of the left ventricle. In some cases, depending on the functional defects of these cells, additional malformations are found in the expected spatial migratory area of the cells, namely in the pharyngeal arch derivatives and cervico-facial structures. Associated non-cardiovascular anomalies are often underestimated, since the multipotency and functional alteration of these cells can result in the modification of multiple neural, epidermal, and cervical structures at different levels. In most cases, patients do not display the full phenotype of abnormalities, but congenital cardiac defects involving the ventricular outflow tract, ascending aorta, aortic arch and supra-aortic trunks should be considered as markers for possible impaired function of these cells. Neural crest cells should not be considered as a unique cell population but on the basis of their cervical rhombomere origins R3-R5 or R6-R7-R8 and specific migration patterns: R3-R4 towards arch II, R5-R6 arch III and R7-R8 arch IV and VI. A better understanding of their development may lead to the discovery of unknown associated abnormalities, thereby enabling potential improvements to be made to the therapeutic approach