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Hepatocytes grown in a dish are immature and do not metabolize compounds as a real liver would. Here, the authors supply stem cell-derived hepatocytes with amino acids at a higher concentration than nutritionally necessary, changing the metabolism of these cells, making them more mature and useful for drug screening and toxicity studies.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by progressive bone marrow failure (BMF) during childhood, aside from numerous congenital abnormalities. FA mouse models have been generated; however, they do not fully mimic the hematopoietic phenotype. As there is mounting evidence that the hematopoietic impairment starts already in utero, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanisms underlying BMF in FA and its developmental basis. Using zinc finger nuclease (ZFN) technology, we have created a knockout of FANCA in human embryonic stem cells (hESC). We introduced a selection cassette into exon 2 thereby disrupting the FANCA coding sequence and found that whereas mono-allelically targeted cells retain an unaltered proliferation potential, disruption of the second allele causes a severe growth disadvantage. As a result, heterogeneous cultures arise due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC are pursued either through selection or single cell cloning, this rapidly results in growth arrest and such cultures cannot be maintained. These data highlight the importance of a functional FA pathway at the pluripotent stem cell stage.

Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process.

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10-20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development.

Summary: The procedures used routinely for collagen and lipofuscin evaluation are, in many cases, qualitative, observer dependent, and disregard spatial distribution. Here, we present a protocol for automatic quantification and spatial characterization of collagen and lipofuscin from label-free microscopy images of human ventricular tissues. We describe the steps for sample collection, tissue processing, image acquisition, and quantification of collagen and lipofuscin. This protocol avoids discrepancies between observers and can be adapted to other tissues and species.For complete details on the use and execution of this protocol, please refer to García-Mendívil et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Connexin 43 (CX43) is one of the major components of gap junctions, the structures responsible for the intercellular communication and transmission of the electrical impulse in the left ventricle. There is limited information on the histological changes of CX43 with age and their effect on electrophysiology, especially in humans. Here, we analyzed left ventricular biopsies from living donors starting at midlife to characterize age-related CX43 remodeling. We assessed its quantity, degree of lateralization, and spatial heterogeneity together with fibrotic deposition. We observed no significant age-related remodeling of CX43. Only spatial heterogeneity increased slightly with age, and this increase was better explained by biological age than by chronological age. Importantly, we found that CX43 features varied considerably among individuals in our population with no relevant relationship to age or fibrosis content, in contrast to animal species. We used our experimental results to feed computational models of human ventricular electrophysiology and to assess the effects of interindividual differences in specific features of CX43 and fibrosis on conduction velocity, action potential duration, and arrhythmogenicity. We found that larger amounts of fibrosis were associated with the highest arrhythmic risk, with this risk being increased when fibrosis deposition was combined with a reduction in CX43 amount and/or with an increase in CX43 spatial heterogeneity. These mechanisms underlying high arrhythmic risk in some individuals were not associated with age in our study population. In conclusion, our data rule out CX43 remodeling as an age-related arrhythmic substrate in the population beyond midlife, but highlight its potential as a proarrhythmic factor at the individual level, especially when combined with increased fibrosis.

Summary: Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age-associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics.

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs.

Amyotrophic lateral sclerosis (ALS) leads to selective loss of motor neurons. Using motor neurons derived from induced pluripotent stem cells from patients with ALS and FUS mutations, the authors demonstrate that axonal transport deficits that are observed in these cells can be rescued by HDAC6 inhibition.

Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrPC), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrPSc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48–72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrPSc post-inoculation, as determined by Western blotting and ELISA. However, the PrPSc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrPSc post-inoculation, as determined by ELISA, but this PrPSc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrPSc.

Cardiomyocytes’ geometry and connexin 43 (CX43) amount and distribution are structural features that play a pivotal role in electrical conduction. Their quantitative assessment is of high interest in the study of arrhythmias, but it is usually hampered by the lack of automatic tools. In this work, we propose a software algorithm (Myocyte Automatic Retrieval and Tissue Analyzer, MARTA) to automatically detect myocytes from fluorescent microscopy images of cardiac tissue, measure their morphological features and evaluate the expression of CX43 and its degree of lateralization. The proposed software is based on the generation of cell masks, contouring of individual cells, enclosing of cells in minimum area rectangles and splitting of these rectangles into end-to-end and middle compartments to estimate CX43 lateral-to-total ratio. Application to human ventricular tissue images shows that mean differences between automatic and manual methods in terms of cardiomyocyte length and width are below 4 μm. The percentage of lateral CX43 also agrees between automatic and manual evaluation, with the interquartile range approximately covering from 3% to 30% in both cases. MARTA is not limited by fiber orientation and has an optimized speed by using contour filtering, which makes it run hundreds of times faster than a trained expert. Developed for CX43 studies in the left ventricle, MARTA is a flexible tool applicable to morphometric and lateralization studies of other markers in any heart chamber or even skeletal muscle. This open-access software is available online.

Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

Summary: To understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro. : Verfaillie and colleagues describe the inefficient cortical neuron, but not motorneuron, generation, from FTD-patient-derived iPSCs carrying a mutation in the GRN gene. They show restoration of the defective phenotype following introduction of the GRN cDNA in FTD-iPSC using zinc finger nucleases and by inhibiting the WNT pathway.