CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples

Generation of American mink induced pluripotent stem cells: a protocol

Mammalian genome reprogramming has been studied for more than half a century. First, Sir John Gurdon showed the possibility of differentiated cell genome reprogramming by enucleated oocyte factors in 1962. Dr. Shinya Yamanaka produced induced pluripotent stem (iPS) cells from mouse fibroblasts by the use of just four transcription factors in 2006: Oct4, Klf4, Sox2, and c-Myc. Generation of iPS cells put a question about the reprogramming completeness: do genes derived from fibroblasts retain their expression? And are the features of iPS cells in compliance with those of embryonic stem (ES) cells that serve as a standard? To date, iPS cells have been produced for tens of species, while ES cells, for less than twenty. In 1993 American mink (Neovison vison) ES cells were produced in the Institute of Cytology and Genetics SB RAS. That created a unique opportunity for comparison of induced and embryo-derived pluripotent cells. In 2015 we produced American mink iPS cells and showed fibro-blast genome reprogramming at the level of gene expression and divided genes into four groups: reprogrammed, with intermediate expression, non-reprogrammed, and the ones with a “novel” expression pattern. Thus, an opportunity to study pluripotency and differentiation on two pluripotent cell types, ES and iPS cells, was added for one more species. In this article we present a detailed protocol for generation of American mink iPS cells with human OCT4, KLF4, SOX2, and c­MYC genes. In addition, we briefly describe necessary methods for their analysis: morphology, cytogenetic analysis, PCR with reverse transcription for the presence of pluripotency “marker” genes, and teratoma formation test in immunodeficient mice. This protocol allows reliable and efficient generation of American mink iPS cells from embryonic fibroblasts

Germline-restricted chromosome (GRC) is widespread among songbirds

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages

Efficient chimeric mouse production using a novel embryonic stem cell line

Embryonic stem cells are commonly used for generation of transgenic mice. Embryonic stem cells could participate in the development of chimeric animals after injection into a blastocyst. Injection of genetically modified embryonic stem cells could lead to germ line transmission of a transgene or genomic modification in chimeric mice. Such founders are used to produce transgenic lines of mice. There are several projects dedicated to production of knock-out mouse lines (KOMP Repository, EUCOMM, Lexicon Genetics). Never-theless, there is a need for complex genome modifications, such as large deletions, reporter genes insertion into the 3’ gene regulatory sequence, or site-specific modifications of the genome. To do that, researchers need an embryonic stem cell line that is able to participate in chimeric animal formation even after prolonged culture in vitro. Several lines of mouse embryonic stem cells were produced in the Laboratory of Developmental Genetics of the Institute of Cytology and Genetics SB RAS. We tested DGES1 cell line (2n = 40, XY) (129S2/SvPasCrl genetic background) for chimeric mice production at the Center for Genetic Resources of Laboratory Animals at ICG SB RAS. Embryonic stem cells were injected into 136 blastocysts (B6D2F1 genetic background), which were transplanted into CD-1 mice. Among 66 progeny, 15 were chimeric, 4 of which were more than 80 % chimeric judged by coat color. All chimeras were males without developmental abnormalities. 10 of 15 males were fertile. Microsatellite analysis of the progeny of chimeric mice revealed embryonic stem cell line DGES1 contribution to the gamete formation. Thus, a novel DGES1 embryonic stem cell line could be efficiently used for transgenic mouse production using B6D2F1 blastocysts and CD-1 recipients

Ultrastructural Abnormalities in Induced Pluripotent Stem Cell-Derived Neural Stem Cells and Neurons of Two Cohen Syndrome Patients

Cohen syndrome is an autosomal recessive disorder caused by VPS13B (COH1) gene mutations. This syndrome is significantly underdiagnosed and is characterized by intellectual disability, microcephaly, autistic symptoms, hypotension, myopia, retinal dystrophy, neutropenia, and obesity. VPS13B regulates intracellular membrane transport and supports the Golgi apparatus structure, which is critical for neuron formation. We generated induced pluripotent stem cells from two patients with pronounced manifestations of Cohen syndrome and differentiated them into neural stem cells and neurons. Using transmission electron microscopy, we documented multiple new ultrastructural changes associated with Cohen syndrome in the neuronal cells. We discovered considerable disturbances in the structure of some organelles: Golgi apparatus fragmentation and swelling, endoplasmic reticulum structural reorganization, mitochondrial defects, and the accumulation of large autophagosomes with undigested contents. These abnormalities underline the ultrastructural similarity of Cohen syndrome to many neurodegenerative diseases. The cell models that we developed based on patient-specific induced pluripotent stem cells can serve to uncover not only neurodegenerative processes, but the causes of intellectual disability in general