Embryo Model Research Articles

Cynomolgus monkey embryo model captures gastrulation and early pregnancy.

Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential is limited. Here, we construct cynomolgus monkey blastoids resembling blastocysts in morphology and transcriptomics using naive ESCs. These blastoids develop to embryonic disk with the structures of yolk sac, chorionic cavity, amnion cavity, primitive streak, and connecting stalk along the rostral-caudal axis through prolonged invitro culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk sac endoderm, three germ layers, and hemato-endothelial progenitors in IVC cynomolgus monkey blastoids were observed by single-cell transcriptomics or immunostaining. Moreover, transferring cynomolgus monkey blastoids to surrogates achieves pregnancies, as indicated by progesterone levels and presence of early gestation sacs. Our results reveal the capacity of invitro gastrulation and invivo early pregnancy of cynomolgus monkey blastoids, providing a useful system to dissect primate embryonic development without the same ethical concerns and access challenges in human embryo study.

Block Copolymer Micelles Encapsulating Au(III) Bis(Dithiolene) Complexes as Promising Nanostructures with Antiplasmodial Activity.

Block copolymer micelles (BCMs) can be used to improve the solubility of lipophilic drugs and increase their circulation half-life. Hence, BCMs assembled from MePEG-b-PCL were evaluated as drug delivery systems of gold(III) bis(dithiolene) complexes (herein AuS and AuSe) to be employed as antiplasmodial drugs. These complexes exhibited remarkable antiplasmodial activity against liver stages of the Plasmodiumberghei parasite, and low toxicity in a model of zebrafish embryos. To improve the complexes' solubility, BCMs were loaded with AuS, AuSe, and the reference drug primaquine (PQ). PQ-BCMs (Dh = 50.9 ± 2.8 nm), AuSe-BCMs (Dh = 87.1 ± 9.7 nm), and AuS-BCMs (Dh = 72.8 ± 3.1 nm) were obtained with a loading efficiency of 82.5%, 55.5%, and 77.4%, respectively. HPLC analysis and UV-Vis spectrophotometry showed that the compounds did not suffer degradation after encapsulation in BCMs. In vitro release studies suggest that AuS/AuSe-BCMs present a more controlled release compared with PQ-loaded BCMs. The antiplasmodial hepatic activity of the drugs was assessed in vitro and results indicate that both complexes present higher inhibitory activity than PQ, although encapsulated AuS and AuSe presented lower activity than their non-encapsulated counterparts. Nevertheless, these results suggest that the use of BCMs as delivery vehicles for lipophilic metallodrugs, particularly AuS and AuSe, could enable the controlled release of complexes and improve their biocompatibility, constituting a promising alternative to conventional antimalarial treatments.

Folic Acid Attenuates MSG-Induced Teratogenicity during A 2-Month Pregnancy by Preventing Neural Crest Cell Destruction and Malformation in Chick Embryo Models

Monosodium glutamate (MSG), commonly used as a food enhancer, has been reported to have teratogenicity during the first 3 days of development. Furthermore, the neural crest cells (NCCs) are crucial for embryonic development during organogenesis. The present study aimed to investigate the treatment effect of folic acid (FA) on MSG-induced teratogenicity, focusing on the toxicity and teratogenic effects on somatic and neural crest cells in chick embryos as models. Six hundred and fifty fertilized eggs were divided into control, FA, MSG, and MSG with FA groups. The chemicals were administered, and the results were investigated after 3 days of incubation. The morphology and histology were studied using stereomicroscopy and hematoxylin and eosin staining, respectively. The NCC population was confirmed by the presence of HNK-1 using immunohistochemistry. The finding showed that the MSG at 2 mg/kg of egg weight induced retardation, tissue malformation, craniofacial, and heart defects, whereas the FA alleviated those adverse effects and reduced the MSG-induced NCCs destruction in the eyes, heart, stomach, and nerves. In conclusion, although MSG harms embryos, FA effectively diminished its teratogenicity in the chick embryo model. These experimental protocols are beneficial for teratogenic studies on preventing birth defects that are harmful to the embryo. HIGHLIGHTS The 4 mg/kg egg weight of monosodium glutamate exposure leads to chick embryo lethality Folic acid treatment is effective at 2 mg/kg of monosodium glutamate exposure Folic acid is essential for supervised neural crest cell functions following monosodium glutamate-induced teratogenesis These experimental protocols are beneficial for teratogenic studies GRAPHICAL ABSTRACT

Assessment of Biocompatibility of 3D‐Printed Cardiovascular Stent

AbstractThis study aims to evaluate the biocompatibility of the 3D‐printed stent and its efficiency on reducing the oxidized‐low‐density lipoprotein (ox‐LDL) damage in endothelial cells using the zebrafish embryos model. The human umbilical vein endothelial cells (HUVECs) significantly (p‐value <0.001) lose their viability upon exposure to ox‐LDL treatment (100 µg/ml). An exposure of HUVECs to resveratrol (RSL)‐unloaded 3D stent slightly lowers the cell viability as compared with untreated control group. On the contrary, RSL‐loaded 3D stent exposure groups maintain initial cell viability. The amounts of TNF‐α and IL‐β released from zebrafish embryos (1.82 ± 0.75 and 1.87 ± 0.24) in the group treated with the RSL‐loaded 3D stents are significantly lower (p < 0.001) than those from the LPS alone treated group (8.13 ± 2.29 and 23.79 ± 3.82, respectively). It is found that RSL‐loaded 3D stent at a RSL dose of 1 mm shows no mortality during the developmental stages, but RSL‐loaded 3D stent at a dose of 2 mm shows a lowered survival rate (93.3 ± 3.3%), shorter length of larvae, pericardial and yolk sac edemas of 53.3 ± 23.3% and 3.3 ± 3.3%, respectively. The RSL‐loaded 3D stents efficiently downregulate the proinflammatory cytokines, and protect an organism against oxidative stress, while producing minimal developments defects in zebrafish embryos.

Design of a new self-assembling antioxidant nanomedicine to ameliorate oxidative stress in zebrafish embryos.

Oxidative stress, which is a persistent state of elevated reactive oxygen species (ROS), is implicated in the pathogeneses of several diseases, making antioxidant-based therapeutics the aptest intervention. Nevertheless, the clinical failure of conventional low-molecular-weight (LMW) antioxidants in oxidative stress-related diseases to yield favorable therapeutic outcomes and an increased mortality rate attributable to their poor pharmacokinetic characteristics, necessitates the development of alternative therapeutics. In light of this, we designed and synthesized a new amphiphilic polymer functionalized with a clinically safe base polymer of poly(styrene-co-maleic anhydride) copolymer conjugated with the LMW pleiotropic antioxidant TEMPO (a potent antioxidant) and biocompatible poly(ethylene glycol) (TEMPO-installed PSMA-g-PEG), which self-assembles into nano-sized micelles (SMAPoTN) under physiological conditions. We investigated its safety and antioxidant ability using zebrafish models. Common LMW antioxidants, such as 4-hydroxy-TEMPO (TEMPOL), vitamin C, N-acetyl-L-cysteine, and edaravone exposure induced phenotypic distortions, a manifestation of developmental toxicity, and resulted in high lethality in zebrafish larvae. LMW TEMPOL also adversely affected embryo hatchability, induced arrhythmia and cardiac edema, and failed to protect against oxidative stress. In contrast, exposure of zebrafish embryos to SMAPoTN increased the hatchability, protected embryos against various inducers of oxidative stress, and did not induce any phenotypic alterations or discernible toxicity. Taken together, we conclude that SMAPoTN surpasses LMW TEMPOL in terms of the ability to protect zebrafish, attributable to efficient ROS scavenging without perturbing normal redox homeostasis. These results imply that SMAPoTN can be used as a therapeutic intervention against various oxidative stress-induced diseases. STATEMENT OF SIGNIFICANCE: Failure of low molecular weight (LMW) antioxidants to improve therapeutic index in various oxidative stress-related pathogenesis, attributable to their poor pharmacokinetic characteristics, greatly limits their clinical translation. To overcome this limitation, we developed a self-assembling antioxidant nanoparticle (SMAPoTN) comprised of amphiphilic polymer; poly(styrene-co-maleic anhydride) conjugated with TEMPO as an antioxidant and biocompatible poly(ethylene glycol). Preliminary studies carried out in the in vivo models of zebrafish embryos confirmed that exposure of LMW antioxidant resulted in acute developmental toxicity, high lethality, and failure to rescue embryos against oxidative stress inducers. In contrast, SMAPoTN did not exert discernible toxicity and significantly improved their survival under oxidative stress. Our finding establishes antioxidant nanoparticles as more suitable therapeutic intervention for oxidative stress-induced diseases than LMW antioxidants.

Correction to: 'Behind the developing brains and beating hearts of stem cell-derived embryo models' (2023) by Amadei and Glover.

Open AccessMoreSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail Cite this article Amadei Gianluca and Glover David M. 2023Correction to: ‘Behind the developing brains and beating hearts of stem cell-derived embryo models’ (2023) by Amadei and GloverOpen Biol.13230034230034http://doi.org/10.1098/rsob.230034SectionOpen AccessCorrectionCorrection to: ‘Behind the developing brains and beating hearts of stem cell-derived embryo models’ (2023) by Amadei and Glover Gianluca Amadei Gianluca Amadei Google Scholar Find this author on PubMed Search for more papers by this author and David M. Glover David M. Glover Google Scholar Find this author on PubMed Search for more papers by this author Gianluca Amadei Gianluca Amadei Google Scholar Find this author on PubMed Search for more papers by this author and David M. Glover David M. Glover Google Scholar Find this author on PubMed Search for more papers by this author Published:08 March 2023https://doi.org/10.1098/rsob.230034This article corrects the followingResearch ArticleBehind the developing brains and beating hearts of stem cell-derived embryo modelshttps://doi.org/10.1098/rsob.220325 Gianluca Amadei and David M. Glover volume 13issue 1Open Biology11 January 2023 Review history Review history is available via Web of Science at https://www.webofscience.com/api/gateway/wos/peer-review/10.1098/rsob.230034/ Open Biology13, 220325. (Published online 11 January 2023.) (https://doi.org/10.1098/rsob.220325)The conflict of interest disclosures should have read as follows:Conflict of interest declaration. D.M.G. is a member of the Open Biology Editorial Board and is the spouse of Magdalena Zernicka-Goetz. As an author of the above Commentary article, D.M.G. had no involvement in the peer review or editorial decision-making, which was handled by Subject Editor Marek Mlodzik. G.A. declares no conflicts of interest.This has been corrected on the publisher's website. Previous Article VIEW FULL TEXT DOWNLOAD PDF FiguresRelatedReferencesDetailsRelated articlesBehind the developing brains and beating hearts of stem cell-derived embryo models11 January 2023Open Biology This IssueMarch 2023Volume 13Issue 3 Article InformationDOI:https://doi.org/10.1098/rsob.230034PubMed:36883284Published by:Royal SocietyOnline ISSN:2046-2441History: Manuscript received06/02/2023Manuscript accepted06/02/2023Published online08/03/2023 License:© 2023 The Authors.Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Citations and impact Subjectsdevelopmental biology

Developmental changes in lipid and fatty acid metabolism and the inhibition by in ovo feeding oleic acid in Muscovy duck embryogenesis.

Hepatic lipid and fatty acid (FA) metabolism are critical for regulating energetic homeostasis during embryogenesis. At present, it remains unclear how an exogenous FA intervention affects embryonic development in an avian embryo model. In Exp. 1, 30 fertilized eggs were sampled on embryonic days (E) 16, 19, 22, 25, 28, 31 and the day of hatch (DOH) to determine the critical period of lipid metabolism. In Exp. 2, a total of 120 fertilized eggs were divided into two groups (60 eggs/group) for in ovo feeding (IOF) procedures on E25. Eggs were injected into the yolk sac with PBS as the control group and with oleic acid (OA) as the IOF-OA treatment group. Samples were collected on E28 and E31. In Exp. 1, hepatic triacylglycerol (TG) and cholesterol (CHO) contents increased while serum TG content decreased from E16 to DOH (P<0.05). Both serum and liver displayed an increase in unsaturated FA and a decrease in saturated FA (P<0.05). There was a quadratic increase in the target gene and protein expression related to hepatic FA de novo synthesis and oxidation (P<0.05), whose inflection period was between E22 and E28. In Exp. 2, compared with the control embryos, IOF-OA embryos had an increased yolk sac TG content on E28 and E31, and a decreased serum TG and CHO content on E28 (P<0.05). The IOF-OA embryos had less OA in the yolk sac and liver on E28, and less unsaturated FA in the serum and liver on E31 than did the control embryos (P<0.05). Hepatic gene mRNA expression related to FA uptake, synthesis, and oxidation on E28 was lower in IOF-OA than in control embryos (P<0.05), not on E31 (P>0.05). Maximal metabolic changes in lipid and FA metabolism occurred on E22-E28 in Muscovy duck embryogenesis, along with the altered target gene and protein expression related to lipogenesis and lipolysis. IOF-OA intervention on E25 could inhibit the target gene expression related to FA uptake, synthesis, and oxidation, which may influence the normal FA metabolism on E28 during embryogenesis.

Novel SMAD3 variant identified in a patient with familial aortopathy modeled using a zebrafish embryo assay.

In human, pathogenic variants in smad3 are one cause of familial aortopathy. We describe a novel SMAD3 variant of unknown significance (VUS), V244F, in a patient who presented with aortic root dilation, right coronary artery ectasia, abdominal aortic aneurysm, right vertebral artery atresia, and cavernoma. Determination of variant pathogenicity impacted multiple aspects of the patient's care, including the most appropriate surgical threshold for which to recommend a valve-sparing aortic root replacement. To determine whether the newly identified SMAD3 variant, and whether SMAD3 induced aortopathy in general, can be assayed in a zebrafish embryo model, we injected smad3a mRNA into Tg[kdrl:mCherry] zebrafish embryos. By measuring the size of the dorsal aorta at 48hpf we found a correlation between pathogenic SMAD3 variants and increased dorsal aortic diameter. The newly identified V244F variant increased dorsal aortic diameter (p < 0.0001) similar to that of the pathogenic control variant T261I (p < 0.0084). In addition, we examined several previously identified variants of uncertain significance and found P124T (p < 0.0467), L296P (p < 0.0025) and A349P (p < 0.0056) to behave like T261I. These results demonstrate that the zebrafish embryo assay was successful in validating known pathogenic variants, classifying our newly identified variant V244F as likely pathogenic, and classifying previously identified variants P124T, L296P, and A349P as likely pathogenic. Overall, our findings identify a novel SMAD3 variant that is likely pathogenic as well as offer a new mechanism to model SMAD3 VUSs in vivo.

Generating human blastoids modeling blastocyst-stage embryos and implantation.

Human early development sets the stage for embryonic and adult life but remains difficult to investigate. A solution came from the ability of stem cells to organize into structures resembling preimplantation embryos-blastocysts-that we termed blastoids. This embryo model is available in unlimited numbers and could thus support scientific and medical advances. However, its predictive power depends on how faithfully it recapitulates the blastocyst. Here, we describe how we formed human blastoids that (1) efficiently achieve the morphology of the blastocyst and (2) form lineages according to the pace and sequence of blastocyst development, (3) ultimately forming cells that transcriptionally reflect the blastocyst (preimplantation stage). We employ three different commercially available 96- and 24-well microwell plates with results similar to our custom-made ones, and show that blastoids form in clinical in vitro fertilization medium and can be cryopreserved for shipping. Finally, we explain how blastoids replicate the directional process of implantation into endometrial organoids, specifically when these are hormonally stimulated. It takes 4 d for human blastoids to form and 10 d to prepare the endometrial implantation assay, and we have cultured blastoids up to 6 d (time-equivalent of day 13). On the basis of our experience, we anticipate that a person with ~1 year of human pluripotent stem cell culture experience and of organoid culture should be able to perform the protocol. Altogether, blastoids offer an opportunity to establish scientific and biomedical discovery programs for early pregnancy, and an ethical alternative to the use of embryos.

Green Synthesis of Blue-Emitting Graphene Oxide Quantum Dots for In Vitro CT26 and In Vivo Zebrafish Nano-Imaging as Diagnostic Probes.

Graphene oxide quantum dots (GOQDs) are prepared using black carbon as a feedstock and H2O2 as a green oxidizing agent in a straightforward and environmentally friendly manner. The process adopted microwave energy and only took two minutes. The GOQDs are 20 nm in size and have stable blue fluorescence at 440 nm. The chemical characteristics and QD morphology were confirmed by thorough analysis using scanning electron microscope (SEM), transmission electron microscope (TEM), atomic force microscope (AFM), Fourier transmission infra-red (FT-IR), and X-ray photoelectron spectroscopy (XPS). The biocompatibility test was used to evaluate the toxicity of GOQDs in CT26 cells in vitro and the IC50 was found to be 200 µg/mL with excellent survival rates. Additional in vivo toxicity assessment in the developing zebrafish (Danio rerio) embryo model found no observed abnormalities even at a high concentration of 400 μg/mL after 96 h post fertilization. The GOQDs luminescence was also tested both in vitro and in vivo. They showed excellent internal distribution in the cytoplasm, cell nucleus, and throughout the zebrafish body. As a result, the prepared GOQDs are expected to be simple and inexpensive materials for nano-imaging and diagnostic probes in nanomedicine.

Qilong Capsule Alleviated MPTP-Induced Neuronal Defects by Inhibiting Apoptosis, Regulating Autophagy in Zebrafish Embryo Model.

Qilong capsule (QLC) originates from the famous "Buyang Huanwu decoction" prescription. It is representative of drugs used in China during recovery from stroke, but its neuroprotective mechanism of action remains obscure. HPLC was used to evaluate the similarity of 10 batches of QLC samples. Then we used a zebrafish model to study the neuroprotective effect of QLC. At 24 hpf, embryos were treated with QLC and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and zebrafish were observed the neuronal length and the number of apoptotic cells in the brain at 72 hpf. At 120 hpf, we conduct zebrafish behavioural tests. We then also used qPCR to detect the expression of genes related to autophagy and apoptosis. The results showed that QLC significantly reduced the damage of dopaminergic neurons, the number of apoptotic cells in the brain, and alleviated motor disturbances induced by MPTP. We found that the mechanism of QLC activity involved decreased neuron cell death by inhibiting mitochondrial apoptosis and autophagy, promoting autophagy, degradation of alpha-synuclein, and neuron cell growth, and rescuing the function of neurons damaged by MPTP. The results indicated that QLC protected against MPTP-induced neuron injury and provided pharmacological evidence for clinical use of QLC.

Comparative assessment of embryotoxicity of 2,4,6-triiodophenol to mouse blastoid and pre-implantation embryo models

Embryonic developmental effects of disinfection by-products, which are generated during drinking water treatment and widely detected in environment, have gained more and more attention nowadays, calling for construction of in vitro research models which can mimic early embryonic development to evaluate the embryotoxicity. The embryonic stem cell test offers a promising assay to predict embryotoxicity of environmental pollutions. However, it is not appropriate for the toxicological study of preimplantation embryos. Here, we used mouse extended stem cells (mEPS) to reconstruct embryo-like structures (blastoid), furtherly attempting to evaluate the reliability of this model for the prediction of possible developmental toxicity of 2,4,6-triiodophenol (TIP, 5–50 μM), a novel halogenated disinfection byproduct widely detected in water and even drinking water, to mammalian preimplantation embryo. To verify this, we treated mouse embryo derived from in vitro fertilization (IVF-embryo) as reference. The results showed that mEPS-blastoid was like natural blastocyst in morphology, cell composition, and could recapitulate key developmental events happened during mouse preimplantation stage. When blastoid and IVF-embryo models were separately exposed to TIP, their final blastocyst formation rates were not impaired, according to morphological features, meanwhile that TIP exposure caused slight cell apoptosis. Besides, TIP induced an ICM cell bias in cell fate decision, resulting in cell proportion change, which implied abnormal developmental potential. Though we could not evaluate TIP’s embryotoxicity before 8-cell stage using blastoid model, its viability as a novel and high-throughput assessment platform for increasing environmental pollutants was still recognized.

Toxicity and teratogenicity investigation of methanolic bark extract of Terminalia arjuna (Roxb.) wight and arn in zebrafish (Danio rerio) embryo model

The present research work aims to establish the teratogenic effect of the Terminalia arjuna (T.arjuna) bark extract using fish embryos through zebrafish embryo model. In the present study, selected zebrafish embryos were exposed to a range of methanolic extract of T.arjuna bark and the changes the embryos undergo were identified by comparing them with a control group. The embryo toxicity was assessed by observing morphological abnormalities after 24, 48, 72 and 96 h exposure and end teratogenicity points were assessed after 48 h exposure. All the embryos were imaged and analyzed for various developmental anomalies such as coagulated embryos, lack of somite formation, non-detachment of the tail, and lack of heartbeat, using imageJ software. From the results obtained, it was found that the embryos exposed with 10 mg/L of T.arjuna showed regular developmental patterns similar to control up to 72 h 5 % mortality was observed post 72 h period. 50 and 100 mg/L showed 100 % mortality after 96 h and 72 h respectively. At 48 h, Embryos treated with 50 mg/L of T.arjuna showed 5.58 % decreased heart beat rate, 7.89 % of decrease in eye diameter decreased head trunk angle of 3.56 % and increased yolk sac width of 0.79 %. After 48 h exposure to 50 and 100 mg/L of T.arjuna extract, 45 % and 85 % of the embryos showed the teratogenic endpoint of coagulation respectively with bent tail. Increased yolk sac width was observed in 100 mg/L treated embryos with 1.01 % after 24 h, also at 48 h decreased heart beat rate of 4.02 %,16.8 % of increased eye diameter, with decreased head trunk angle of 2.35 %, decreased yolk sac width of 5.13 % were observed. After 48 h exposure of T.arjuna bark extract, the morphological, physiological changes and teratogenic end points were eminent in 50 and 100 mg/L treated embryos, thus the results signify that methanolic bark extract of T. arjuna possess teratogenic effects at this concentration in zebrafish embryos.

Metabolic profiling of Ochradenus baccatus Delile. utilizing UHPLC-HRESIMS in relation to the in vitro biological investigations

Ochradenus baccatus Delile (Resedaceae) is a desert plant with edible fruits native to the Middle East. Few investigators have reported antibacterial, antiparasitic and anti-cancer activities of the plant. Herein we evaluated the cytotoxic activity of O. baccatus using four cell lines and a zebrafish embryo model. Additionally, liquid chromatography coupled with mass spectroscopy was performed to characterize the extract’s main constituents. The highest cytotoxicity was observed against human cervical adenocarcinoma (HeLa), with CC50 of 39.1 µg/mL and a selectivity index (SI) of 7.23 (p < 0.01). Metabolic analysis of the extract resulted in the annotation of 57 metabolites, including fatty acids, flavonoids, glucosinolates, nitrile glycosides, in addition to organic acids. The extract showed an abundance of hydroxylated fatty acids (16 peaks). Further, 3 nitrile glycosides have been identified for the first time in Ochradenus sp., in addition to 2 glucosinolates. These identified phytochemicals may partially explain the cytotoxic activity of the extract. We propose O. baccatus as a possible safe food source for further utilization to partially contribute to the increasing food demand specially in Saharan countries.

Synergy of ruthenium metallo-intercalator, [Ru(dppz)2(PIP)]2+, with PARP inhibitor Olaparib in non-small cell lung cancer cells

Poly(ADP-ribose) polymerase (PARP) are critical DNA repair enzymes that are activated as part of the DNA damage response (DDR). Although inhibitors of PARP (PARPi) have emerged as small molecule drugs and have shown promising therapeutic effects, PARPi used as single agents are clinically limited to patients with mutations in germline breast cancer susceptibility gene (BRCA). Thus, novel PARPi combination strategies may expand their usage and combat drug resistance. In recent years, ruthenium polypyridyl complexes (RPCs) have emerged as promising anti-cancer candidates due to their attractive DNA binding properties and distinct mechanisms of action. Previously, we reported the rational combination of the RPC DNA replication inhibitor [Ru(dppz)2(PIP)]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine, PIP = 2-(phenyl)-imidazo[4,5-f][1,10]phenanthroline), “Ru-PIP”, with the PARPi Olaparib in breast cancer cells. Here, we expand upon this work and examine the combination of Ru-PIP with Olaparib for synergy in lung cancer cells, including in 3D lung cancer spheroids, to further elucidate mechanisms of synergy and additionally assess toxicity in a zebrafish embryo model. Compared to single agents alone, Ru-PIP and Olaparib synergy was observed in both A549 and H1975 lung cancer cell lines with mild impact on normal lung fibroblast MRC5 cells. Employing the A549 cell line, synergy was confirmed by loss in clonogenic potential and reduced migration properties. Mechanistic studies indicated that synergy is accompanied by increased double-strand break (DSB) DNA damage and reactive oxygen species (ROS) levels which subsequently lead to cell death via apoptosis. Moreover, the identified combination was successfully able to inhibit the growth of A549 lung cancer spheroids and acute zebrafish embryos toxicity studies revealed that this combination showed reduced toxicity compared to single-agent Ru-PIP.

Differential Angiogenic Induction Impacts Nasal Polyp Tissue Growth.

In chronic rhinosinusitis with nasal polyps, inflammatory edema drives tissue remodeling favoring anomalous growth of the nasal mucosa, but a proangiogenic contribution of nasal polyp in support of tissue growth is still controversial. The chorioallantoic membrane of chicken embryo model was employed to address the potentiality of nasal tissue fragments to modulate angiogenesis. Fifty-seven fertilized eggs were implanted with polyp or healthy nasal mucosa tissue or were kept as non-implanted controls. The embryos' size, length, and development stage, and chorioallantoic membrane vasculature morphology were evaluated after 48h. Quantitative computer vision techniques applied to digital chorioallantoic membrane images automatically calculated the branching index as the ratio between the areas of the convex polygon surrounding the vascular tree and the vessels' area. Ethics approval and consent to participate: the study was approved by the Human Research Ethics Committee of the Federal University of São Paulo (CAAE number: 80763117.1.0000.5505) and by the Animal Research Ethics Committee of University of São Paulo (nº CEUA 602-2019). Mucosal, but not polyp tissue implants, hampered embryo development and induced underdeveloped chorioallantoic membranes with anastomosed, interrupted, and regressive vessels. Vessels' areas and branching indexes were higher among the chorioallantoic membranes with polyp implants and controls than among those with healthy mucosa implants. Nasal polyp presents differential angiogenic induction that impacts tissue growth.

In vitro and in vivo investigation of chrysin chelated copper complex as biocompatible materials for bone tissue engineering applications.

Flavonoid metal complexes have interesting properties and are widely explored for bone regeneration owing to their potent biological activity. In the present study, we investigated the biocompatibility and osteogenic properties of the Copper(II)-chrysin complex (C/Cu). The biocompatibility of C/Cu was assessed in vitro with human osteoblastic cells and in vivo using chick embryo and zebrafish models. The C/Cu complex was found to be cytofriendly with good biocompatibility. The osteogenic property of C/Cu was studied at cellular and molecular levels. C/Cu promoted mineralization in osteoblastic cultures by increasing ALP activity. At the molecular level, C/Cu significantly promoted the mRNA levels of osteoblast differentiation marker genes such as runt-related transcription factor 2 (Runx2), Type 1 collagen and ALP. In addition to this, secretory proteins, osteonectin (ON) and osteocalcin (OC) levels were also stimulated. We have also identified that C/Cu exhibited enhanced osteogenic properties and antibacterial activity compared with Chrysin. Thus, C/Cu can be used as an osteogenic agent in bone tissue engineering.

Progress in the use of human pluripotent stem cells for in vitro modeling of leprosy and cutaneous tuberculosis

Compared with mature cells, human pluripotent stem cells (PSCs) have cellular and organ functions closer to those of primary cells. In infectious skin disease research, PSCs provide accurate pathophysiology for exploring the specific cellular and molecular mechanisms of refractory disease. Herein, we review the latest knowledge of human embryo model construction under laboratory conditions, focusing on important mycobacterial diseases, such as leprosy and tuberculosis. Moreover, an analysis of SARS‐CoV‐2 research using human PSCs or organoids shows that human PSCs play an important role in modelling dermatological infectious diseases and their complex mechanisms.

Modeling policy development: examining national governance of stem cell-based embryo models.

Researchers can now coax human pluripotent stem cells to imitate the structure and spontaneous self-organization of the developing human embryo. Although these stem cell-based embryo models present an advantageous alternative to embryo research, they also raise ethical and policy challenges. In 2021, the International Society for Stem Cell Research revised its Guidelines for Stem Cell Research and Clinical Translation, providing contemporaneous best practices for ethical conduct in the field. The Guidelines complement national governance frameworks; however, they also contain contentious and aspirational norms that might catalyze change in research practice and in the enactment of national policies. Using a sample of 11 research-intensive countries, the authors compare research policy frameworks against the International Society for Stem Cell Research Guidelines to showcase how developments in global and national policies might affect stem cell-based embryo model research governance and illustrate fertile areas for ethical reflection and policy development.

Evaluation of Stem-Cell Embryo Models by Integration with a Human Embryo Single-Cell Transcriptome Atlas.

Single-cell RNA sequencing (scRNA-seq) revolutionized our understanding of the molecular processes of early development and provided us with the means to capture biological heterogeneity and assess the cellular composition in early embryos. Comparative analysis of the transcriptional landscapes of embryos with single-cell resolution allows us to better understand and improve stem-cell-based embryo models. However, proper comparison between different single-cell datasets acquired by different laboratories and through different technologies is imperative for adequate analysis and findings. In this chapter, we focus on the analysis of human blastoids, which model the blastocyst, and their integrative analysis with human embryo datasets and a 2D in vitro early development model system dataset, which models epiblast, extraembryonic mesoderm, and trophoblast cells.