Search

Background: The cause of duodenal atresia (DA) is not known. Tandler's "solid cord" hypothesis conflicts with current biological evidence. In humans, a genetic aetiology is supported by the association with Trisomy 21. Interruption of Fgf10 is the strongest genetic link to DA in mice, demonstrating an increased incidence and severity as embryos mature. This project aimed to develop an organoid model to facilitate ex vivo DA research on the FGF10/FGFR2b signalling pathway. We hypothesised that DA morphology represents an evolving spectrum of disease and that Fgf10 knockout organoids would vary in growth pattern compared to wild-type., Methods: Organoids were cultured from the duodenum of E12.5 Fgf10 knockout, heterozygous and wild-type embryos, using an air-liquid interface with Growth Factor reduced Matrigel. Organoids were photographed every 48 h to observe growth. Organoids were isolated and fixed after 14 days, then stained with DAPI, KI-67, and cytokeratin to demonstrate proliferation and differentiation., Results: Wild-type duodenum developed into crypt-forming organoids. Fgf10 heterozygous duodenum failed to progress beyond the development stage of spheroids. Fgf10 knockout duodenum failed to demonstrate any growth. Wholemount staining showed the greatest cell proliferation and differentiation in wild-type tissue., Conclusion: This research presents a novel concept for the growth of embryonic gastrointestinal tissue to inform normal biology. The small sample numbers and restricted culture duration limit longer-term growth analysis. While this model serves as a potential ex vivo setting for future research, that research should consider organoid models with greater standardisation and other gastrointestinal regions., Level of Evidence: Animal/laboratory study., Competing Interests: Conflicts of interest The authors of this paper have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hematopoietic stem cells (HSCs) derived from human induced pluripotent stem cells (iPS cells) have important biomedical applications. We identified differentiation conditions that generate HSCs defined by robust long-term multilineage engraftment in immune-deficient NOD,B6.Prkdc scid Il2rg tm1Wjl/SzJ Kit W41/W41 mice. We guided differentiating iPS cells, as embryoid bodies in a defined culture medium supplemented with retinyl acetate, through HOXA-patterned mesoderm to hemogenic endothelium specified by bone morphogenetic protein 4 and vascular endothelial growth factor (VEGF). Removal of VEGF facilitated an efficient endothelial-to-hematopoietic transition, evidenced by release into the culture medium of CD34 + blood cells, which were cryopreserved. Intravenous transplantation of two million thawed CD34 + cells differentiated from four independent iPS cell lines produced multilineage bone marrow engraftment in 25-50% of immune-deficient recipient mice. These functionally defined, multipotent CD34 + hematopoietic cells, designated iPS cell-derived HSCs (iHSCs), produced levels of engraftment similar to those achieved following umbilical cord blood transplantation. Our study provides a step toward the goal of generating HSCs for clinical translation., (© 2024. The Author(s).)

Phosphorylated histone H2AX (γH2AX) represents a sensitive molecular marker of DNA double-strand breaks (DSBs) and is implicated in stem cell biology. We established a model of mouse embryonic stem cell (mESC) differentiation and examined the dynamics of γH2AX foci during the process. Our results revealed high numbers of γH2AX foci in undifferentiated mESCs, decreasing as the cells differentiated towards the endothelial cell lineage. Notably, we observed two distinct patterns of γH2AX foci: the typical discrete γH2AX foci, which colocalize with the transcriptionally permissive chromatin mark H3K4me3, and the less well-characterized clustered γH2AX regions, which were only observed in intermediate progenitor cells. Next, we explored responses of mESCs to γ-radiation ( 137 Cs). Following exposure to γ-radiation, mESCs showed a reduction in cell viability and increased γH2AX foci, indicative of radiosensitivity. Despite irradiation, surviving mESCs retained their differentiation potential. To further exemplify our findings, we investigated neural stem progenitor cells (NSPCs). Similar to mESCs, NSPCs displayed clustered γH2AX foci associated with progenitor cells and discrete γH2AX foci indicative of embryonic stem cells or differentiated cells. In conclusion, our findings demonstrate that γH2AX serves as a versatile marker of DSBs and may have a role as a biomarker in stem cell differentiation. The distinct patterns of γH2AX foci in differentiating mESCs and NSPCs provide valuable insights into DNA repair dynamics during differentiation, shedding light on the intricate balance between genomic integrity and cellular plasticity in stem cells. Finally, the clustered γH2AX foci observed in intermediate progenitor cells is an intriguing feature, requiring further exploration., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our previous study showing the successful lung deposition of aerosolised palivizumab in lambs, this current study evaluated the "proof-of-principle" effect of aerosolised palivizumab delivered as a therapeutic to neonatal lambs following RSV infection. (2) Methods: Neonatal lambs were intranasally inoculated with RSV-A2 on day 0 (day 3 post-birth) and treated with aerosolised palivizumab 3 days later (day 3 post-inoculation). Clinical symptoms, RSV viral load and inflammatory response were measured post-inoculation. (3) Results: Aerosolised therapeutic delivery of palivizumab did not reduce RSV viral loads in the nasopharynx nor the bronchoalveolar lavage fluid, but resulted in a modest reduction in inflammatory response at day 6 post-inoculation compared with untreated lambs. (4) Conclusions: This proof-of-principle study shows some evidence of aerosolised palivizumab reducing RSV inflammation, but further studies using optimized protocols are needed in order to validate these findings.

Background/purpose: What causes normal descent of the testis in a fetus, and what goes wrong with this complex process to cause undescended testes (UDT), or cryptorchidism? Over the last 2 decades, most authors searching for the cause(s) of UDT have looked at the 2 main hormones involved, insulin-like hormone 3 (Insl3) and testosterone (T)/ dihydrotestosterone (DHT), and their known upstream (hypothalamic-pituitary axis) and intracellular 'downstream' pathways. Despite these detailed searches, the genetic causes of UDT remain elusive, which suggest the aetiology is multifactorial, and/or we are looking in the wrong place., Methods: In this review we highlight the intricate morphological steps involved in testicular descent, which we propose may contain the currently 'idiopathic' causes of UDT. By integrating decades of research, we have underlined many areas that have been overlooked in the search for causes of UDT., Results: It is quite likely that the common causes of UDT are still hidden in these areas, and we suggest examining these processes is worthwhile in the hope of finding the common genetic anomalies that lead to cryptorchidism. Given the fact that a fibrous barrier preventing descent is often described at orchidopexy, examination of the extracellular matrix enzymes needed to allow gubernacular migration may be a fruitful place to start., Conclusion: This review of the complex anatomical steps and hormonal regulation of testicular descent highlights many areas of morphology and signalling pathways that have been overlooked in the search for causes of UDT., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Androgens control rodent inguinoscrotal testicular descent during a "programming window" (E12-17). It is proposed that androgen masculinises the genitofemoral nerve, but the mechanism remains unknown. We investigate androgen receptor (AR)-containing target organs: inguinal fat pad (IFP) and mammary bud (MB), supplied by the genitofemoral nerve, hypothesizing that neurotrophic factors may retrogradely masculinise the GFN., Methods: The IFP, MB and bulbocavernosus (BC) muscle were collected at E12.5/E17.5 from androgen receptor knockout (ARKO) mice and wild-type (WT) littermates. Immunofluorescence and gene expression (RT-qPCR; n = 8/group) for Bdnf, active (TrkB) and inactive (truncated TrkB) receptors, Cntf and Cntf receptor were performed., Results: In the IFP at E12.5, ARKO TrkB mRNA expression was significantly downregulated compared to WT males (p < 0.0026). By E17.5, there was increased Bdnf expression (p < 0.0233). The MB had no differences at E12.5 and had regressed in WT males by E17.5. The BC had no differences at E12.5, but at E17.5 had significant upregulation of Bdnf expression in ARKO, compared to WT males. There were no differences in CNTF or CNTF receptor expression., Conclusions: Androgen alters active TrkB and Bdnf expression in the IFP. IFP Bdnf signalling may regulate "masculinisation" of the GFN sensory nerves to indirectly control inguinoscrotal testicular descent., Impact: Androgen mediates neurotrophin release in the inguinal fat pad in mice, which may facilitate normal testicular descent by masculinising the GFN by peripheral uptake of neurotrophin. This is the first study to examine the role of neurotrophins in testicular descent. This suggests novel steps in the mechanical process of normal testicular descent that may be abnormal in some children with undescended testes., (© 2021. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Purpose: Postnatal surge of gonadotrophins, Luteinizing hormone (LH) and Follicle-Stimulating hormone (FSH) known as minipuberty, is critical for gonocyte maturation into spermatogonial stem cells (SSC) in the testis. Gonadotrophins are essential for optimum fertility in men, but very little is known how they regulate germ cells during minipuberty. This study examined whether gonadotrophins play a role on gonocyte transformation in vivo., Methods: Testes from hypogonadal (hpg) mice and their wild type (WT) littermates (n = 6/group) were weighed, and processed in paraffin at postnatal days (D) 0, 3, 6 and 9. Mouse VASA homologue (germ cell marker), anti-Müllerian hormone (Sertoli cell marker) antibodies and DAPI (nuclei marker) were used for immunofluorescence followed by confocal imaging. Germ cells on or off basement membrane (BM) and Sertoli cells/tubule were counted using Image J and analyzed with GraphPad., Results: Comparing to WT littermates, there were significantly fewer germ cells on BM/tubule (p < 0.05) in D9 hpg mice, whereas there was no significant difference for germ cells off BM/tubule and Sertoli cells/tubule between littermates. However, testicular weight was significantly reduced in D3-D9 hpg mice comparing to WT littermates., Conclusion: Gonadotrophin deficiency reduced D9 germ cells on BM indicating impaired gonocyte transformation into SSC. This suggests that gonadotrophins may mediate gonocyte transformation during minipuberty.

Purpose: Undescended Testes (UDT) are prevalent in 2%-5% of male infants and cause malignancy and infertility. During germ cell development, abnormal gonocytes usually undergo apoptosis. This process is believed to involve BAX (Bcl-2 Associated X) protein in clearing abnormal gonocytes which may fail in UDT, resulting in persisting gonocytes causing seminomas later in life. We aim to investigate the role of BAX in gonocyte apoptosis., Materials and Methods: BAXKO (BAX-knockout) mice were back-crossed to OG2 mice (Oct4-promoter driving enhanced green fluorescent protein-eGFP) to produce BAXOG2 mice. Testes (wildtype-BAX +/+ , heterozygous-BAX +/- and homozygous-BAX -/- mice, n = 6/group) on postnatal days 1, 3, 6, 9 were fixed and embedded in OCT for frozen sectioning. Sections were labeled with Anti-Müllerian Hormone (Sertoli cell marker), Mouse Vasa Homolog (germ cell marker) and DAPI (nucleus marker) and imaged using confocal microscopy. Oct4-GFP+ve germ cells, germ cells on/off the basement membrane and Sertoli cells were counted using ImageJ followed by data analysis with GraphPad., Results: BAX -/- OG2 mice had significantly higher number of germ cells/tubule comparing to BAX +/+ OG2 on day 9. There were Oct4-GFP+ve gonocyte-like germ cells that persisted in the center of the tubules in BAX -/- OG2 even after the completion of gonocyte transformation. This suggests that abnormal gonocytes in BAX -/- OG2 mice failed to undergo apoptosis and are allowed to persist., Conclusion: This study demonstrated that apoptosis is important in regulating germ cell migration and differentiation during gonocyte transformation in neonatal mice. In addition, inhibition of apoptosis results in persisting neonatal gonocytes which might become seminomas in patients with UDT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The inguinoscrotal stage of testicular descent is characterized by an increase in cell density and collagen fibers as the gubernaculum undergoes cell division and increases Extracellular Matrix (ECM) activity. Rats that lack the enzyme Adamts16, a known ECM proteinase, develop cryptorchidism postnatally and are infertile. Therefore, this study aims to investigate the link between the Adamts16 enzyme and congenital undescended testes (UDT) in Adamts16 knockout (KO) rats during postnatal development., Methods: Formalin-fixed specimens of Wild-Type, Adamts16 heterozygous and Adamts16 homozygous KO rats post birth were sectioned and used for standard H&E histology and Masson's trichrome staining. A quantitative analysis on image J was performed to determine the intensity of collagen fibers within the inguinoscrotal fat pad (IFP) (n = 3 age/genotype)., Results: The migration of the gubernaculum within the Adamts16 heterozygous and Adamts16 KO rat was considerably disrupted. Furthermore, the Masson's trichrome staining demonstrated a significant increase in collagen fibers around the gubernaculum of rats that lacked Adamts16 enzyme at day 8., Conclusion: This study reports a failure of gubernacular migration leading to UDT in Adamts16 KO rats during development, suggesting that the expression of Adamts16 gene is critical for normal gubernacular migration through the breakdown of collagen fibers within the IFP., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: Duodenal atresia (DA) is a congenital bowel obstruction requiring major surgery in the first week of life. Three morphological phenotypes are described, reflecting increasing degrees of obstruction and discontinuity of the duodenum. The cause of DA is not known. Tandler's original "solid cord" hypothesis conflicts with recent biological evidence, and is unable to account for differing DA types. In humans, a genetic etiology is supported by the association between Trisomy 21 and DA, and reports of familial inheritance patterns. Interruption of FGF10/FGFR2b signaling is the best demonstrated genetic link to DA in mice, with 35-75% of homozygous knockout embryos developing DA., Purpose: This review examines the current evidence surrounding the etiology of DA. We focus on research regarding FGF10/FGFR2b signaling and its role in duodenal and other intestinal atresia. Further, we outline planned future research in this area, that we consider necessary to validate and better understand this murine model in order to successfully translate this research into clinical practice., Conclusion: Determining the etiology of DA in humans is a clinical and scientific imperative. Fgf10/Fgfr2b murine models represent current science's best key to unlocking this mystery. However, further research is required to understand the complex role of FGF10/FGFR2b signaling in DA development. Such complexity is expected, given the lethality of their associated defects makes ubiquitous interruption of either Fgf10 or Fgfr2b genes an unlikely cause of DA in humans. Rather, local or tissue-specific mutation in Fgf10 , Fgfr2b , or their downstream targets, is the hypothesized basis of DA etiology., (Copyright © 2020 Jones, Sarila, Chapuis, Hutson, King and Teague.)

In the heart, primary microRNA-208b (pri-miR-208b) and Myheart (Mhrt) are long non-coding RNAs (lncRNAs) encoded by the cardiac myosin heavy chain genes. Although preclinical studies have shown that lncRNAs regulate gene expression and are protective for pathological hypertrophy, the mechanism underlying sex-based differences remains poorly understood. In this study, we examined DNA- and RNA-methylation-dependent regulation of pri-miR-208b and Mhrt. Expression of pri-miR-208b is elevated in the left ventricle of the female heart. Despite indistinguishable DNA methylation between sexes, the interaction of MeCP2 on chromatin is subject to RNase digestion, highlighting that affinity of the methyl-CG reader is broader than previously thought. A specialized procedure to isolate RNA from soluble cardiac chromatin emphasizes sex-based affinity of an MeCP2 co-repressor complex with Rest and Hdac2. Sex-specific Mhrt methylation chromatinizes MeCP2 at the pri-miR-208b promoter and extends the functional relevance of default transcriptional suppression in the heart., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Duodenal atresia (DA) is a congenital obstruction of the duodenum, which affects 1 in 7000 pregnancies and requires major surgery in the 1st days of life. Three morphological DA types are described. In humans, the association between DA and Down syndrome suggests an underlying, albeit elusive, genetic etiology. In mice, interruption of fibroblast growth factor 10 ( Fgf10 ) gene signaling results in DA in 30-50% of embryos, supporting a genetic etiology. This study aims to validate the spectrum of DA in two novel strains of Fgf10 knock-out mice, in preparation for future and translational research. Methods: Two novel CRISPR Fgf10 knock-out mouse strains were derived and embryos generated by heterozygous plug-mating. E15.5-E19.5 embryos were genotyped with respect to Fgf10 and micro-dissected to determine the presence and type of DA. Results: One twenty seven embryos (32 wild-type, 34 heterozygous, 61 null) were analyzed. No wild-type or heterozygous embryos had DA. However, 74% of Fgf10 null embryos had DA (49% type 1, 18% type 2, and 33% type 3). Conclusion: Our CRISPR-derived strains showed higher penetrance of DA due to single-gene deletion of Fgf10 in mice than previously reported. Further, the DA type distribution in these mice more closely reiterated that observed in humans. Future experiments will document RNA and protein expression of FGF10 and its key downstream signaling targets in normal and atretic duodenum. This includes exploitation of modern, high-fidelity developmental tools, e.g., Fgf10 flox/+ -tomato flox/flox mice.