Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells.

Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks.

[For a good understanding and use of the term "organoids"]. / Pour une bonne compréhension et un bon usage du terme « organoïdes ¼.

Title: Pour une bonne compréhension et un bon usage du terme « organoïdes ¼. Abstract: Depuis une dizaine d'années, des progrès considérables ont été réalisés concernant les conditions qui permettent à des cellules de s'auto-organiser dans l'espace comme elles le font lors des phases précoces du développement embryonnaire ou dans certains tissus adultes. On nomme ainsi « organoïdes ¼ des structures en trois dimensions complexes, organisées et intégrant plusieurs types cellulaires, qui peuvent reproduire in vitro certaines fonctions d'un organe. Toutefois, ces organoïdes ne peuvent actuellement reproduire à l'identique une architecture anatomique et fonctionnelle complète. Bien qu'utilisé pour des raisons de simplification pour la communication, en particulier dans la presse généraliste, il est donc abusif d'utiliser le terme « mini-organes ¼ pour décrire ces structures.

Successful 3D imaging of cleared biological samples with light sheet fluorescence microscopy.

In parallel with the development of tissue-clearing methods, over the last decade, light sheet fluorescence microscopy has contributed to major advances in various fields, such as cell and developmental biology and neuroscience. While biologists are increasingly integrating three-dimensional imaging into their research projects, their experience with the technique is not always up to their expectations. In response to a survey of specific challenges associated with sample clearing and labeling, image acquisition, and data analysis, we have critically assessed the recent literature to characterize the difficulties inherent to light sheet fluorescence microscopy applied to cleared biological samples and to propose solutions to overcome them. This review aims to provide biologists interested in light sheet fluorescence microscopy with a primer for the development of their imaging pipeline, from sample preparation to image analysis. Importantly, we believe that issues could be avoided with better anticipation of image analysis requirements, which should be kept in mind while optimizing sample preparation and acquisition parameters.

Deciphering neuronal deficit and protein profile changes in human brain organoids from patients with creatine transporter deficiency.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3ß, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3ß, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.

Three-dimensional molecular cartography of human cerebral organoids revealed by double-barcoded spatial transcriptomics.

Spatially resolved transcriptomics is revolutionizing our understanding of complex tissues, but scaling these approaches to multiple tissue sections and three-dimensional tissue reconstruction remains challenging and cost prohibitive. In this work, we present a low-cost strategy for manufacturing molecularly double-barcoded DNA arrays, enabling large-scale spatially resolved transcriptomics studies. We applied this technique to spatially resolve gene expression in several human brain organoids, including the reconstruction of a three-dimensional view from multiple consecutive sections, revealing gene expression heterogeneity throughout the tissue.

Human Brain Organoids-on-Chip: Advances, Challenges, and Perspectives for Preclinical Applications.

There is an urgent need for predictive in vitro models to improve disease modeling and drug target identification and validation, especially for neurological disorders. Cerebral organoids, as alternative methods to in vivo studies, appear now as powerful tools to decipher complex biological processes thanks to their ability to recapitulate many features of the human brain. Combining these innovative models with microfluidic technologies, referred to as brain organoids-on-chips, allows us to model the microenvironment of several neuronal cell types in 3D. Thus, this platform opens new avenues to create a relevant in vitro approach for preclinical applications in neuroscience. The transfer to the pharmaceutical industry in drug discovery stages and the adoption of this approach by the scientific community requires the proposition of innovative microphysiological systems allowing the generation of reproducible cerebral organoids of high quality in terms of structural and functional maturation, and compatibility with automation processes and high-throughput screening. In this review, we will focus on the promising advantages of cerebral organoids for disease modeling and how their combination with microfluidic systems can enhance the reproducibility and quality of these in vitro models. Then, we will finish by explaining why brain organoids-on-chips could be considered promising platforms for pharmacological applications.

iPSCs derived from infertile men carrying complex genetic abnormalities can generate primordial germ-like cells.

Despite increasing insight into the genetics of infertility, the developmental disease processes remain unclear due to the lack of adequate experimental models. The advent of induced pluripotent stem cell (iPSC) technology has provided a unique tool for in vitro disease modeling enabling major advances in our understanding of developmental disease processes. We report the full characterization of complex genetic abnormalities in two infertile patients with either azoospermia or XX male syndrome and we identify genes of potential interest implicated in their infertility. Using the erythroblasts of both patients, we generated primed iPSCs and converted them into a naive-like pluripotent state. Naive-iPSCs were then differentiated into primordial germ-like cells (PGC-LCs). The expression of early PGC marker genes SOX17, CD-38, NANOS3, c-KIT, TFAP2C, and D2-40, confirmed progression towards the early germline stage. Our results demonstrate that iPSCs from two infertile patients with significant genetic abnormalities are capable of efficient production of PGCs. Such in vitro model of infertility will certainly help identifying causative factors leading to early germ cells development failure and provide a valuable tool to explore novel therapeutic strategies.

[Chimeric embryos and pseudo-embryos: An alternative to human embryos for research]. / Des embryons chimères et des pseudo-embryons comme alternatives pour la recherche sur l'embryon humain.

The study of human development is essential to further our knowledge and to improve our therapeutic strategies in the fields of reproductive and regenerative medicine. Given the limited access to supernumerary embryos and the prohibition on creating new ones for research, two alternative strategies can be proposed to study human embryonic development. The first is to create pseudo-embryos or blastoids. The second is to create human/animal chimeric embryos by injecting pluripotent stem cells, ES or iPS, into animal embryos. We explain herein the importance of these new experimental paradigms for studying human development and their complementarity.

TITLE: Des embryons chimères et des pseudo-embryons comme alternatives pour la recherche sur l'embryon humain. ABSTRACT: L'étude du développement humain est indispensable afin d'approfondir nos connaissances et, à long terme, perfectionner nos stratégies thérapeutiques dans les domaines de la médecine de la reproduction et de la médecine régénératrice. Face à la limite d'accès aux embryons surnuméraires et à l'interdiction d'en créer de nouveaux seulement à des fins de recherche, deux stratégies alternatives peuvent être proposées pour étudier le développement embryonnaire humain. La première consiste à fabriquer des pseudo-embryons ou blastoïdes. La seconde consiste à créer des embryons chimères homme/animal par injection de cellules souches pluripotentes, ES ou iPS, dans des embryons d'animaux. Nous expliquons ici l'importance de ces nouveaux paradigmes expérimentaux pour étudier le développement humain, et leur complémentarité.

Long Term Gene Expression in Human Induced Pluripotent Stem Cells and Cerebral Organoids to Model a Neurodegenerative Disease.

Human brain organoids (mini-brains) consist of self-organized three-dimensional (3D) neural tissue which can be derived from reprogrammed adult cells and maintained for months in culture. These 3D structures manifest substantial potential for the modeling of neurodegenerative diseases and pave the way for personalized medicine. However, as these 3D brain models can express the whole human genetic complexity, it is critical to have access to isogenic mini-brains that only differ in specific and controlled genetic variables. Genetic engineering based on retroviral vectors is incompatible with the long-term modeling needed here and implies a risk of random integration while methods using CRISPR-Cas9 are still too complex to adapt to stem cells. We demonstrate in this study that our strategy which relies on an episomal plasmid vector derived from the Epstein-Barr virus (EBV) offers a simple and robust approach, avoiding the remaining caveats of mini-brain models. For this proof-of-concept, we used a normal tau protein with a fluorescent tag and a mutant genetic form (P301S) leading to Fronto-Temporal Dementia. Isogenic cell lines were obtained which were stable for more than 30 passages expressing either form. We show that the presence of the plasmid in the cells does not interfere with the mini-brain differentiation protocol and obtain the development of a pathologically relevant phenotype in cerebral organoids, with pathological hyperphosphorylation of the tau protein. Such a simple and versatile genetic strategy opens up the full potential of human organoids to contribute to disease modeling, personalized medicine and testing of therapeutics.

Proof-of-Concept Study of Drug Brain Permeability Between in Vivo Human Brain and an in Vitro iPSCs-Human Blood-Brain Barrier Model.

The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.

Small-molecule induction of Aß-42 peptide production in human cerebral organoids to model Alzheimer's disease associated phenotypes.

Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aß), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aß accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-ß Forty-Two Inducer named Aftin-5. Secreted, soluble Aß fragment concentrations were analyzed in MB-conditioned media. An increase in the Aß42 fragment secretion was observed as was an increased Aß42/Aß40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aß accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aß concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aß oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.

Induced pluripotent stem cell generation from a man carrying a complex chromosomal rearrangement as a genetic model for infertility studies.

Despite progress in human reproductive biology, the cause of male infertility often remains unknown, due to the lack of appropriate and convenient in vitro models of meiosis. Induced pluripotent stem cells (iPSCs) derived from the cells of infertile patients could provide a gold standard model for generating primordial germ cells and studying their development and the process of spermatogenesis. We report the characterization of a complex chromosomal rearrangement (CCR) in an azoospermic patient, and the successful generation of specific-iPSCs from PBMC-derived erythroblasts. The CCR was characterized by karyotype, fluorescence in situ hybridization and oligonucleotide-based array-comparative genomic hybridization. The CCR included five breakpoints and was caused by the inverted insertion of a chromosome 12 segment into the short arm of one chromosome 7 and a pericentric inversion of the structurally rearranged chromosome 12. Gene mapping of the breakpoints led to the identification of a candidate gene, SYCP3. Erythroblasts from the patient were reprogrammed with Sendai virus vectors to generate iPSCs. We assessed iPSC pluripotency by RT-PCR, immunofluorescence staining and teratoma induction. The generation of specific-iPSCs from patients with a CCR provides a valuable in vitro genetic model for studying the mechanisms by which chromosomal abnormalities alter meiosis and germ cell development.

Creating conditions for the success of the French industrial advanced therapy sector.

Although the European Union merely followed the initiatives of the United States and Japan by introducing special regimes for orphan medicinal products, it has introduced a special status for a new category of biological medicinal products, advanced therapy medicinal products (ATMPs), adopting specific associated regulations. European Regulation (which constitutes the highest legal instrument in the hierarchy of European law texts) [EC] No. 1394/2007, published in 2007, uses this term to define somatic cell therapy medicinal products, tissue-engineered products, and gene therapy medicinal products, possibly combined with medical devices. The stated objective was two-fold: both to promote their industrialization and market access, while guaranteeing a high level of health protection for patients. Since publication of the regulation, few marketing authorizations have been granted in Europe, and these have not been accompanied by commercial success. However, certain recent studies show that this is a growing sector and that France remains the leading European nation in terms of clinical trials. This round table brought together a panel of representatives of French public and private protagonists from the advanced therapy sector. The discussions focused on the conditions to ensure the success of translational research and, more generally, the French advanced therapy sector. These enabled a number of obstacles to be identified, which once lifted, by means of recommendations, would facilitate the development and success of this sector.

Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model.

BACKGROUND: Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. DESIGN AND METHODS: We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. RESULTS: We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. CONCLUSIONS: These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment.

Partial reversal of the methylation pattern of the X-linked gene HUMARA during hematopoietic differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) can be induced to differentiate towards hematopoiesis with high efficiency. In this work, we analyzed the methylation status of the X-linked HUMARA (human androgen receptor) gene in hematopoietic cells derived from hESC line H9 before and after induction of hematopoietic differentiation. All passages of H9 and H9-derived hematopoietic cells displayed homogenous methylation pattern with disappearance of the same allele upon HpaII digestion. This pattern persisted in the great majority of different hematopoietic progenitors derived from H9, except in 11 of 86 individually plucked colonies in which an equal digestion of the HUMARA alleles has been found, suggesting that a methylation change occurring at this locus during differentiation. Interestingly, quantification of X inactive-specific transcript (XIST) RNA in undifferentiated H9 cell line and day 14 embryoid bodies (EB) by RT-PCR did not show any evidence of XIST expression either before or after differentiation. Thus, during self-renewal conditions and after induction of commitment towards the formation of EB, the methylation pattern of the HUMARA locus appears locked with the same unmethylated allele. However, hematopoietic differentiation seems to be permissive to the reversal of methylation status of HUMARA in some terminally differentiated progenitors. These data suggest that monitoring methylation of HUMARA gene during induced differentiation could be of use for studying hESC-derived hematopoiesis.

Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells.

Surface antigens on hematopoietic stem cells (HSCs) enable prospective isolation and characterization. Here, we compare the cell-surface phenotype of hematopoietic repopulating cells from murine yolk sac, aorta-gonad-mesonephros, placenta, fetal liver, and bone marrow with that of HSCs derived from the in vitro differentiation of murine embryonic stem cells (ESC-HSCs). Whereas c-Kit marks all HSC populations, CD41, CD45, CD34, and CD150 were developmentally regulated: the earliest embryonic HSCs express CD41 and CD34 and lack CD45 and CD150, whereas more mature HSCs lack CD41 and CD34 and express CD45 and CD150. ESC-HSCs express CD41 and CD150, lack CD34, and are heterogeneous for CD45. Finally, although CD48 was absent from all in vivo HSCs examined, ESC-HSCs were heterogeneous for the expression of this molecule. This unique phenotype signifies a developmentally immature population of cells with features of both primitive and mature HSC. The prospective fractionation of ESC-HSCs will facilitate studies of HSC maturation essential for normal functional engraftment in irradiated adults.

Enhanced plating efficiency of trypsin-adapted human embryonic stem cells is reversible and independent of trisomy 12/17.

Human embryonic stem cells (hESCs) can be cultured abundantly and indefinitely, but are subject to accumulations of chromosomal aberrations. To preserve their genetic integrity, hESCs are commonly maintained as cell aggregates or clumps during passaging. However, clump passaging hinders large-scale culture and complicates the isolation of single cell clones. To facilitate the isolation of genetically modified clones of hESCs while preserving their genetic integrity, we employed trypsin single-cell passaging for brief periods before returning to clump passaging for long-term maintenance. We observed that accommodation to trypsin passage as single cells is an adaptive process where over three to four passages considerably increases the plating efficiency. However, trypsin passage was associated with abnormalities of chromosomes 12 and 17. Nevertheless, the high plating efficiency of trypsin passaged hESCs is a reversible phenotype, regardless of chromosomal abnormalities, suggesting that epigenetic events are responsible for the switch in phenotype.

The cdx-hox pathway in hematopoietic stem cell formation from embryonic stem cells.

Embryonic stem cells (ESCs) differentiated in vitro will yield a multitude of hematopoietic derivatives, yet progenitors displaying true stem cell activity remain difficult to obtain. Possible causes are a biased differentiation to primitive yolk sac-type hematopoiesis, and a variety of developmental or functional deficiencies. Recent studies in the zebrafish have identified the caudal homeobox transcription factors (cdx1/4) and posterior hox genes (hoxa9a, hoxb7a) as key regulators for blood formation during embryonic development. Activation of Cdx and Hox genes during the in vitro differentiation of mouse ESCs followed by co-culture on supportive stromal cells generates ESC-derived hematopoietic stem cells (HSCs) capable of multilineage repopulation of lethally irradiated adult mice. We show here that brief pulses of ectopic Cdx4 or HoxB4 expression are sufficient to enhance hematopoiesis during ESC differentiation, presumably by acting as developmental switches to activate posterior Hox genes. Insights into the role of the Cdx-Hox gene pathway during embryonic hematopoietic development in the zebrafish have allowed us to improve the derivation of repopulating HSCs from murine ESCs.

Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity.

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation, which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time, attesting to a selective advantage of transduced progenitors. Notably, a high transgene copy number was detected in all lymphoid organs, and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether, these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases.