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Increasing survival rates of children following cancer treatment have resulted in a significant population of adult survivors with the common side effect of infertility. Additionally, the availability of genetic testing has identified Klinefelter syndrome (classic 47,XXY) as the cause of future male infertility for a significant number of prepubertal patients. This study explores new spermatogonia stem cell (SSC)-based fertility therapies to meet the needs of these patients. Testicular cells were isolated from cryopreserved human testes tissue stored from XY and XXY prepubertal patients and propagated in a two-dimensional culture. Cells were then incorporated into a 3D human testicular organoid (HTO) system. During a 3-week culture period, HTOs maintained their structure, viability, and metabolic activity. Cell-specific PCR and flow cytometry markers identified undifferentiated spermatogonia, Sertoli, Leydig, and peritubular cells within the HTOs. Testosterone was produced by the HTOs both with and without hCG stimulation. Upregulation of postmeiotic germ cell markers was detected after 23 days in culture. Fluorescence in situ hybridization (FISH) of chromosomes X, Y, and 18 identified haploid cells in the in vitro differentiated HTOs. Thus, 3D HTOs were successfully generated from isolated immature human testicular cells from both euploid (XY) and Klinefelter (XXY) patients, supporting androgen production and germ cell differentiation in vitro.

IntroductionPlacenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing.MethodsWe used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene “lcoET3” into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs).ResultsPLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 “machinery”, resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness.DiscussionAlthough we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.

Abstract Compounds that induce 5‐aminolevulinic acid [ALA] synthase‐1 and/or cytochromes P‐450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence‐based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase‐1 [ALAS‐1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS‐1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, β‐estradiol, hydroxychloroquine sulfate, alpha‐methyldopa, D (‐) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi‐well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

IntroductionEnvironmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future.MethodsWe employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate), and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM–10 mM), mercury (200 nM–200 µM), thallium (10 nM–10 µM), or glyphosate (25 µM–25 mM) for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity.ResultsAs expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures.Conclusion3D organoids have significant utility to be deployed in additional toxicity screening applications, such as future development of treatments to mitigate exposures, drug screening, and environmental toxin detection.

The FRAS1-related extracellular matrix 1 (FREM1) gene encodes an extracellular matrix protein that plays a critical role in the development of multiple organ systems. In humans, recessive mutations in FREM1 cause eye defects, congenital diaphragmatic hernia, renal anomalies and anorectal malformations including anteriorly placed anus. A similar constellation of findings-microphthalmia, cryptophthalmos, congenital diaphragmatic hernia, renal agenesis and rectal prolapse-have been described in FREM1-deficient mice. In this paper, we identify a homozygous Frem1 missense mutation (c.1687A>T, p.Ile563Phe) in an N-ethyl-N-nitrosourea (ENU)-derived mouse strain, crf11, with microphthalmia, cryptophthalmos, renal agenesis and rectal prolapse. This mutation affects a highly conserved residue in FREM1's third CSPG domain. The p.Ile563Phe change is predicted to be deleterious and to cause decreased FREM1 protein stability. The crf11 allele also fails to complement the previously described eyes2 allele of Frem1 (p.Lys826*) providing further evidence that the crf11 phenotype is due to changes affecting Frem1 function. We then use mice bearing the crf11 and eyes2 alleles to identify lung lobulation defects and decreased anogenital distance in males as novel phenotypes associated with FREM1 deficiency in mice. Due to phenotypic overlaps between FREM1-deficient mice and mice that are deficient for the retinoic acid-responsive transcription factor GATA4 and the extracellular matrix protein SLIT3, we also perform experiments to look for in vivo genetic interactions between the genes that encode these proteins. These experiments reveal that Frem1 interacts genetically with Gata4 in the development of lung lobulation defects and with Slit3 in the development of renal agenesis. These results demonstrate that FREM1-deficient mice faithfully recapitulate many of the phenotypes seen in individuals with FREM1 deficiency and that variations in GATA4 and SLIT3 expression modulate some FREM1-related phenotypes in mice.