A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles.

Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology. We hypothesised that, based on developments in microfluidics and human organoid technology, in vitro multi-organ-on-a-chip (MOC) models allow us to study effects of sEVs in modelled human organs like kidney and liver in a semi-systemic manner. Human kidney- and liver organoids combined by microfluidic channels maintained physiological functions, and a kidney injury model was established using hydrogenperoxide. MSC-sEVs were isolated, and their size, density and potential contamination were analysed. These sEVs stimulated recovery of the renal epithelium after injury. Microscopic analysis shows increased accumulation of PKH67-labelled sEVs not only in injured kidney cells, but also in the unharmed liver organoids, compared to healthy control conditions. In conclusion, this new MOC model recapitulates therapeutic efficacy and biodistribution of MSC-sEVs as observed in animal models. Its human background allows for in-depth analysis of the MoA and identification of potential side effects.

Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus.

Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.

Immune Reconstitution after Allogeneic Hematopoietic Cell Transplantation in Children.

Allogeneic (allo) hematopoietic cell transplantation (HCT) has evolved into a potent curative treatment option for a variety of malignant and nonmalignant diseases. The occurrence of complications and mortality after allo-HCT is, however, still high and is strongly associated with immune reconstitution (IR). Therefore, detailed information on IR through immunomonitoring is crucial to improve survival chances after HCT. To date, information about the reconstituting immune system after allo-HCT in pediatric patients is mostly derived from routine standard-of-care measurements. More profound knowledge on IR may provide tools to better predict and modulate adverse reactions and, subsequently, improve survival chances. Here, we provide an overview of IR (eg, immune cell subsets and circulating chemokines/cytokines) after allo-HCT in children, taking into account different cell sources and serotherapy, and discuss strategies to enhance immunomonitoring. We conclude that available IR data after allo-HCT contain limited information on immune cell families (mostly only generic T, B, and NK cells), which would improve with more detailed information on reconstituting cell subsets or effector cell functionality at earlier time points (<1 month). In addition, secretome data (eg, multiplex cytokine/chemokine profiles) could add to the understanding of IR mechanisms and cell functionality and may even provide (early) biomarkers for individual disease outcome, such as viral reactivity, graft-versus-host disease, or graft-versus-leukemia. The present data and suggestions for more detailed, standardized, and harmonized immunomonitoring in future (pediatric) allo-HCT studies will pave the path to "precision transplantation:" an individualized HCT approach (including conditioning), based on detailed information on IR and biomarkers, aiming to reduce transplantation related mortality and relapse, and subsequently improve survival chances.