Organoids and chimeras: the hopeful fusion transforming traumatic brain injury research.

Research in the field of traumatic brain injury has until now heavily relied on the use of animal models to identify potential therapeutic approaches. However, a long series of failed clinical trials has brought many scientists to question the translational reliability of pre-clinical results obtained in animals. The search for an alternative to conventional models that better replicate human pathology in traumatic brain injury is thus of the utmost importance for the field. Recently, orthotopic xenotransplantation of human brain organoids into living animal models has been achieved. This review summarizes the existing literature on this new method, focusing on its potential applications in preclinical research, both in the context of cell replacement therapy and disease modelling. Given the obvious advantages of this approach to study human pathologies in an in vivo context, we here critically review its current limitations while considering its possible applications in traumatic brain injury research.

Genome-wide identification and analysis of monocot-specific chimeric jacalins (MCJ) genes in Maize (Zea mays L.).

BACKGROUND: The monocot chimeric jacalins (MCJ) proteins, which contain a jacalin-related lectin (JRL) domain and a dirigent domain (DIR), are specific to Poaceae. MCJ gene family is reported to play an important role in growth, development and stress response. However, their roles in maize have not been thoroughly investigated. RESULTS: In this study, eight MCJ genes in the maize genome (designated as ZmMCJs) were identified, which displayed unequal distribution across four chromosomes. Phylogenetic relationships between the ZmMCJs were evident through the identification of highly conserved motifs and gene structures. Analysis of transcriptome data revealed distinct expression patterns among the ZmMCJ genes, leading to their classification into four different modules, which were subsequently validated using RT-qPCR. Protein structures of the same module are found to be relatively similar. Subcellular localization experiments indicated that the ZmMCJs are mainly located on the cell membrane. Additionally, hemagglutination and inhibition experiments show that only part of the ZmMCJs protein has lectin activity, which is mediated by the JRL structure, and belongs to the mannose-binding type. The cis-acting elements in the promoter region of ZmMCJ genes predicted their involvement response to phytohormones, such as abscisic acid and jasmonic acid. This suggests that ZmMCJ genes may play a significant role in both biotic and abiotic stress responses. CONCLUSIONS: Overall, this study adds new insights into our understanding of the gene-protein architecture, evolutionary characteristics, expression profiles, and potential functions of MCJ genes in maize.

Modeling HBV Infection and Therapy in Immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (FAH) Knockout Mice with Human Chimeric Liver.

Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.

Toward developing human organs via embryo models and chimeras.

Developing functional organs from stem cells remains a challenging goal in regenerative medicine. Existing methodologies, such as tissue engineering, bioprinting, and organoids, only offer partial solutions. This perspective focuses on two promising approaches emerging for engineering human organs from stem cells: stem cell-based embryo models and interspecies organogenesis. Both approaches exploit the premise of guiding stem cells to mimic natural development. We begin by summarizing what is known about early human development as a blueprint for recapitulating organogenesis in both embryo models and interspecies chimeras. The latest advances in both fields are discussed before highlighting the technological and knowledge gaps to be addressed before the goal of developing human organs could be achieved using the two approaches. We conclude by discussing challenges facing embryo modeling and interspecies organogenesis and outlining future prospects for advancing both fields toward the generation of human tissues and organs for basic research and translational applications.

The Effect of Cryopreserved Sperm on the Early Development, Survival, and Growth of Intergeneric Sterbel Hybrids (Acipenser ruthenus × Huso huso).

The aim of this study was to analyze the survival and growth of intergeneric (Acispenser ruthenus × Huso huso L.) sterbel hybrids obtained by fertilizing sterlet eggs with cryopreserved beluga semen. The rate of embryonic development did not differ between sterbel hybrids (experimental groups) and sterlets (control groups), and the hatching period was identical in all groups. The survival rate of hybrid larvae was higher in the experimental groups than in the control groups. Body weight and body length measurements revealed that sterbel hybrids grew at a faster rate than the control group sterlets. The hybrid origin of sterbels produced with the use of cryopreserved beluga semen was confirmed in a genetic analysis based on species-specific DNA fragments. To the best of the authors' knowledge, this is the first study to analyze the growth of sterbel hybrids derived from cryopreserved semen. The research findings indicate that this type of intergeneric hybridization delivers satisfactory results and can be applied in sturgeon aquaculture.

Adaptive substitutions at two amino acids of HCPro modify its functional properties to separately increase the virulence of a potyviral chimera.

We had previously reported that a plum pox virus (PPV)-based chimera that had its P1-HCPro bi-cistron replaced by a modified one from potato virus Y (PVY) increased its virulence in some Nicotiana benthamiana plants, after mechanical passages. This correlated with the natural acquisition of amino acid substitutions in several proteins, including in HCPro at either position 352 (Ile→Thr) or 454 (Leu→Arg), or of mutations in non-coding regions. Thr in position 352 is not found among natural potyviruses, while Arg in 454 is a reversion to the native PVY HCPro amino acid. We show here that both mutations separately contributed to the increased virulence observed in the passaged chimeras that acquired them, and that Thr in position 352 is no intragenic suppressor to a Leu in position 454, because their combined effects were cumulative. We demonstrate that Arg in position 454 improved HCPro autocatalytic cleavage, while Thr in position 352 increased its accumulation and the silencing suppression of a reporter in agropatch assays. We assessed infection by four cloned chimera variants expressing HCPro with none of the two substitutions, one of them or both, in wild-type versus DCL2/4-silenced transgenic plants. We found that during infection, the transgenic context of altered small RNAs affected the accumulation of the four HCPro variants differently and hence, also infection virulence.

Brain Chimeroids reveal individual susceptibility to neurotoxic triggers.

Interindividual genetic variation affects the susceptibility to and progression of many diseases1,2. However, efforts to study how individual human brains differ in normal development and disease phenotypes are limited by the paucity of faithful cellular human models, and the difficulty of scaling current systems to represent multiple people. Here we present human brain Chimeroids, a highly reproducible, multidonor human brain cortical organoid model generated by the co-development of cells from a panel of individual donors in a single organoid. By reaggregating cells from multiple single-donor organoids at the neural stem cell or neural progenitor cell stage, we generate Chimeroids in which each donor produces all cell lineages of the cerebral cortex, even when using pluripotent stem cell lines with notable growth biases. We used Chimeroids to investigate interindividual variation in the susceptibility to neurotoxic triggers that exhibit high clinical phenotypic variability: ethanol and the antiepileptic drug valproic acid. Individual donors varied in both the penetrance of the effect on target cell types, and the molecular phenotype within each affected cell type. Our results suggest that human genetic background may be an important mediator of neurotoxin susceptibility and introduce Chimeroids as a scalable system for high-throughput investigation of interindividual variation in processes of brain development and disease.

Human-mouse chimeric brain models constructed from iPSC-derived brain cells: Applications and challenges.

The establishment of reliable human brain models is pivotal for elucidating specific disease mechanisms and facilitating the discovery of novel therapeutic strategies for human brain disorders. Human induced pluripotent stem cell (iPSC) exhibit remarkable self-renewal capabilities and can differentiate into specialized cell types. This makes them a valuable cell source for xenogeneic or allogeneic transplantation. Human-mouse chimeric brain models constructed from iPSC-derived brain cells have emerged as valuable tools for modeling human brain diseases and exploring potential therapeutic strategies for brain disorders. Moreover, the integration and functionality of grafted stem cells has been effectively assessed using these models. Therefore, this review provides a comprehensive overview of recent progress in differentiating human iPSC into various highly specialized types of brain cells. This review evaluates the characteristics and functions of the human-mouse chimeric brain model. We highlight its potential roles in brain function and its ability to reconstruct neural circuitry in vivo. Additionally, we elucidate factors that influence the integration and differentiation of human iPSC-derived brain cells in vivo. This review further sought to provide suitable research models for cell transplantation therapy. These research models provide new insights into neuropsychiatric disorders, infectious diseases, and brain injuries, thereby advancing related clinical and academic research.

Integrated morphometric and molecular approaches to screen hybrid from wild Labeo rohita and Labeo catla parent populations.

BACKGROUND: Interspecific hybrids of rohu (Labeo rohita) and catla (Labeo catla) are common, especially in India due to constrained breeding. These hybrids must segregate from their wild parents as part of conservational strategies. This study intended to screen the hybrids from wild rohu and catla parents using both morphometric and molecular approaches. METHODS & RESULTS: The carp samples were collected from Jharkhand and West Bengal, India. The correlation and regression analysis of morphometric features are considered superficial but could be protracted statistically by clustering analysis and further consolidated by nucleotide variations of one mitochondrial and one nuclear gene to differentiate hybrids from their parents. Out of 21 morphometric features, 6 were used for clustering analysis that exhibited discrete separation among rohu, catla, and their hybrids when the data points were plotted in a low-dimensional 2-D plane using the first 2 principal components. Out of 40 selected single nucleotide polymorphism (SNP) positions of the COX1 gene, hybrid showed 100% similarity with catla. Concerning SNP similarity of the 18S rRNA nuclear gene, the hybrid showed 100% similarity with rohu but not with catla; exhibiting its probable parental inheritance. CONCLUSIONS: Along with morphometric analysis, the SNP comparison study together points towards strong evidence of interspecific hybridization between rohu and catla, as these hybrids share both morphological and molecular differences with either parent. However, this study will help screen the hybrids from their wild parents, as a strategy for conservational management.

Whole-genome sequences reveal zygotic composition in chimeric twins.

While most dizygotic twins have a dichorionic placenta, rare cases of dizygotic twins with a monochorionic placenta have been reported. The monochorionic placenta in dizygotic twins allows in utero exchange of embryonic cells, resulting in chimerism in the twins. In practice, this chimerism is incidentally identified in mixed ABO blood types or in the presence of cells with a discordant sex chromosome. Here, we applied whole-genome sequencing to one triplet and one twin family to precisely understand their zygotic compositions, using millions of genomic variants as barcodes of zygotic origins. Peripheral blood showed asymmetrical contributions from two sister zygotes, where one of the zygotes was the major clone in both twins. Single-cell RNA sequencing of peripheral blood tissues further showed differential contributions from the two sister zygotes across blood cell types. In contrast, buccal tissues were pure in genetic composition, suggesting that in utero cellular exchanges were confined to the blood tissues. Our study illustrates the cellular history of twinning during human development, which is critical for managing the health of chimeric individuals in the era of genomic medicine.

Debates on humanization of human-animal brain chimeras - are we putting the cart before the horses?

Research on human-animal chimeras have elicited alarms and prompted debates. Those involving the generation of chimeric brains, in which human brain cells become anatomically and functionally intertwined with their animal counterparts in varying ratios, either via xenografts or embryonic co-development, have been considered the most problematic. The moral issues stem from a potential for "humanization" of the animal brain, as well as speculative changes to the host animals' consciousness or sentience, with consequential alteration in the animal hosts' moral status. However, critical background knowledge appears to be missing to resolve these debates. Firstly, there is no consensus on animal sentience vis-à-vis that of humans, and no established methodology that would allow a wholesome and objective assessment of changes in animal sentience resulting from the introduction of human brain cells. Knowledge in interspecies comparative neuropsychology that could allow effective demarcation of a state of "humanization" is also lacking. Secondly, moral status as a philosophical construct has no scientific and objective points of reference. Either changes in sentience or humanization effects would remain unclear unless there are some neuroscientific research grounding. For a bioethical stance based on moral status of human-animal brain chimera to make meaningful contributions to regulatory policies, it might first need to be adequately informed by, and with its arguments constructed, in a manner that are factually in line with the science. In may be prudent for approved research projects involving the generation of human-animal brain chimera to have a mandatory component of assessing plausible changes in sentience.

Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis.

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Exposure of chimaeric embryos to exogenous FGF4 leads to the production of pure ESC-derived mice.

Producing chimaeras constitutes the most reliable method of verifying the pluripotency of newly established cells. Moreover, forming chimaeras by injecting genetically modified embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into the embryo is part of the procedure for generating transgenic mice, which are used for understanding gene function. Conventional methods for generating transgenic mice, including the breeding of chimaeras and tetraploid complementation, are time-consuming and cost-inefficient, with significant limitations that hinder their effectiveness and widespread applications. In the present study, we modified the traditional method of chimaera generation to significantly speed up this process by generating mice exclusively derived from ESCs. This study aimed to assess whether fully ESC-derived mice could be obtained by modulating fibroblast growth factor 4 (FGF4) levels in the culture medium and changing the direction of cell differentiation in the chimaeric embryo. We found that exogenous FGF4 directs all host blastomeres to the primitive endoderm fate, but does not affect the localisation of ESCs in the epiblast of the chimaeric embryos. Consequently, all FGF4-treated chimaeric embryos contained an epiblast composed exclusively of ESCs, and following transfer into recipient mice, these embryos developed into fully ESC-derived newborns. Collectively, this simple approach could accelerate the generation of ESC-derived animals and thus optimise ESC-mediated transgenesis and the verification of cell pluripotency. Compared to traditional methods, it could speed up functional studies by several weeks and significantly reduce costs related to maintaining and breeding chimaeras. Moreover, since the effect of stimulating the FGF signalling pathway is universal across different animal species, our approach can be applied not only to rodents but also to other animals, offering its utility beyond laboratory settings.

The marine sponge, Hymeniacidon sinapium, displays allorecognition of siblings during post-larval settling and metamorphosis to juveniles.

Marine sponges, including the crumb of bread sponge, Hymeniacidon sinapium, display allorejection responses to contact with conspecifics in both experimental and natural settings. These responses have been used to infer immunocompetence in a variety of marine invertebrates. However, larvae and juveniles from several marine sponge species fuse and form chimeras. Some of these chimeras persist, whereas others eventually break down, revealing a period of allogeneic non-responsiveness that varies depending on the species. Alternatively, for H. sinapium, most pairs of sibling post-larvae and juveniles that settle in contact initiate immediate allorecognition and show the same morphological response progression as the adults. This indicates that allorecognition and response occurs during early metamorphosis. Results from H. sinapium and other sponge species, in addition to annotations of sponge genomes, suggest that allorecognition and immunocompetence in sponges are mediated by distinct systems and may become functional at different times during or after metamorphosis for different species. Consequently, allorecognition may not be a good proxy for the onset of immunocompetence.

Emerging Human Pluripotent Stem Cell-Based Human-Animal Brain Chimeras for Advancing Disease Modeling and Cell Therapy for Neurological Disorders.

Human pluripotent stem cell (hPSC) models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms. In particular, hPSC-based human-animal brain chimeras enable the study of human cell pathophysiology in vivo. In chimeric brains, human neural and immune cells can maintain human-specific features, undergo maturation, and functionally integrate into host brains, allowing scientists to study how human cells impact neural circuits and animal behaviors. The emerging human-animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease, elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels, and testing the efficacy of cell therapy interventions. Here, we discuss recent advances in the generation and applications of using human-animal chimeric brain models for the study of neurological disorders, including disease modeling and cell therapy.