In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Assessing motile cilia coverage and beat frequency in mammalian in vitro cell culture tissues.

Cilia density, distribution and beating frequency are important properties of airway epithelial tissues. These parameters are critical in diagnosing primary ciliary dyskinesia and examining in vitro models, including those derived from induced pluripotent stem cells. Video microscopy can be used to characterize these parameters, but most tools available at the moment are limited in the type of information they can provide, usually only describing the ciliary beat frequency of very small areas, while requiring human intervention and training for their use. We propose a novel and open-source method to fully characterize cilia beating frequency and motile cilia coverage in an automated fashion without user intervention. We demonstrate the ability to differentiate between different coverage densities, identifying even small patches of cilia in a larger field of view, and to fully characterize the cilia beating frequency of all moving areas. We also show that the method can be used to combine multiple fields of view to better describe a sample without relying on small pre-selected regions of interest. This is released with a simple graphical user interface for file handling, enabling a full analysis of individual fields of view in a few minutes on a typical personal computer.

Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells.

RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells.

Global Perspective on the Development of Genetically Modified Immune Cells for Cancer Therapy.

Since the first genetically-engineered clinical trial was posted to clinicaltrials.gov in 2003 (NCT00019136), chimeric antigen receptor (CAR) and T-cell receptor (TCR) therapies have exhibited unprecedented growth. USA, China, and Europe have emerged as major sites of investigation as many new biotechnology and established pharmaceutical companies invest in this rapidly evolving field. Although initial studies focused primarily on CD19 as a target antigen, many novel targets are now being evaluated. Next-generation genetic constructs, starting materials, and manufacturing strategies are also being applied to enhance efficacy and safety and to treat solid tumors as well as hematologic malignancies. Fueled by dramatic clinical efficacy and recent regulatory approvals of CD19-targeted CAR cell therapies, the field of engineered cell therapeutics continues to expand. Here, we review all 745 genetically modified CAR and TCR clinical trials with anticipated accrual of over 28,000 patients posted to clinicaltrials.gov until 31st of December 2019. We analyze projected patient enrollment, geographic distribution and phase of studies, target antigens and diseases, current strategies for optimizing efficacy and safety, and trials expected to yield important clinical data in the coming 6-12 months.

[Beware of immunoglobulin's passive transfer when interpreting serological analyses!]. / Interprétation des tests sérologiques : ne pas oublier le transfert passif des immunoglobulines !

Due to their extended indications, intravenous immunoglobulins (IVIg) are increasingly used in hospital setting. After injection, classical IVIg half-life reaches more than 3weeks. IVIg result from the pooling of many blood donations and contain all natural antibodies usually found in the general population. Administered antibodies are known to interfere with many diagnostic assays, particularly those used for infectious serology. It is not recommended to perform serological determination after IVIg infusion. It is recommended to keep a delay of at least 4months after IVIg infusion before doing any serological assay; failure to do so will result in misinterpretation of biological findings. Interpretation of any serological test after IVIg administration should be particularly cautious.