Morphological Characterization of Astrocytes in a Xenograft of Human iPSCDerived Neural Precursor Cells.

Transplantation of a mixed astrocyte and neuron culture is of interest in the development of cell therapies for neurodegenerative diseases. In this case, an assessment of engraftment requires a detailed morphological characterization, in particular an analysis of the neuronal and glial populations. In the experiment performed, human iPSC-derived neural progenitors transplanted into a rat striatum produced a mixed neuron and astrocyte population in vivo by the sixth month after transplantation. The morphological characteristics and neurochemical profile of the xenografted astrocytes were similar to those of mature human astroglia. Unlike neurons, astrocytes migrated to the surrounding structures and the density and pattern of their distribution in the striatum and cerebral cortex differed, which indicates that the microenvironment affects human glia integration. The graft was characterized by the zonal features of glial cell morphology, which was a reflection of cell maturation in the central area, glial shaft formation around the transplanted neurons, and migration to the surrounding structures.

[Changes of a2-macroglobulin activity and endothelin-1 concentration in tears of rabbits after transplantation of retinal pigment epithelium cells derived from the induced pluripotent stem cells]. / Izmenenie urovnia a2-makroglobulina i éndotelina-1 v sleznoi zhidkosti krolikov posle transplantatsii kletok retinal'nogo pigmentnogo épiteliia, differentsirovannykh iz indutsirovannykh pliuripotentnykh stvolovykh kletok.

Retinal diseases accompanied with the dysfunction or death of the retinal pigment epithelial (RPE) cells are widespread, hard to treat, and appear to be a leading case of visual loss and blindness among the persons older than 55 years. Transplantation of RPE cells derived from the induced pluripotent stem cells (IPSC-RPE) is a promising method of therapy for these diseases. To ensure the transplant survival instant follow-up is required. It can be based on biochemical analyses of tear fluid that can be easily non-invasively collected. For the post-transplantation process monitoring we have choosen such polyfunctional bioregulators as α2-macroglobulin (α2-MG) and endothelin-1 (ET-1). RPE atrophy in New Zealand Albino rabbits was modeled via the subretinal injection of bevacizumab. IPSC-RPE in suspension or as a monolayer on the scaffold were transplanted subretinally 1 month after the injection. α2-MG activity and ET-1 concentration in tears were estimated during the first month and after 2, 3 and 7 months after transplantation. On the 7-14 days after transplantation α2-MG activity increased in tears of the both operated and controlateral eye probably as a reaction on the corticosteroid therapy. In 50% rabbits there was one more increase after 2-3 months that could be due to the immune inflammation. Concentration of ET-1 in tears decreased dramatically on the 7-14 days and 7 months after transplantation, and it could have an influence upon the retinal vassal tone. The data obtained show that estimation of bioregulators in tears can help monitoring local metabolic processes after RPE transplantation that is necessary for the opportune, reasonable and focused medicamental correction of post-transplantation process.

[Experimental stem cell replacement transplantation in retinal pigment epithelium atrophy]. / Zamestitel'naya transplantatsiya stvolovymi kletkami pri atrofii retinal'nogo pigmentnogo epiteliya v eksperimente.

PURPOSE: To develop and evaluate the results of the modified surgical technique for transplantation of retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells (iPSC-RPE) in the form of a cell suspension into the subretinal space of rabbits with previously induced RPE atrophy. MATERIAL AND METHODS: The study was conducted on 10 New Zealand albino rabbits (20 eyes). One month after modeling RPE atrophy and retinal degeneration, rabbits were subjected to subretinal transplantation of iPSC-RPE cells in the form of a cell suspension. To prevent reflux of iPSC-RPE into the vitreal cavity, the injection site was sealed with 2-3 drops of autologous platelet-rich plasma (PRP). All rabbits underwent spectral optical coherence tomography (SOCT) and autofluorescence studies on the Heidelberg Spectralis system («Heidelberg Engineering¼, Germany). Enucleated animal eyes were studied with morphological and immunohistochemical methods. RESULTS: In this study we developed and evaluated a modified surgical technique of transplantation of iPSC-RPE in the form of a cell suspension into the subretinal space of rabbits with induced RPE atrophy. It was found that the use of PRP helps seal the defect and prevents cell suspension reflux into the vitreous cavity, effectively minimizing intra- and postoperative complications. Morphological in vivo study and examination of histological sections showed that implantable iPSC-RPEs were correctly integrated and adhered to the choroid in the surgery site. Immunohistochemical analysis involving fluorescence-marked antibodies confirmed the survival of iPSC-RPE integrated into the retina of model animals. CONCLUSION: This method improves the technology of iPSC-RPE transplantation on preclinical stages of the study, revealing new prospects in the treatment of degenerative diseases of the retina and the possibility of a personalized approach.

Generation of two iPSC lines from healthy donor with a heterozygous mutation in the VPS13B gene.

The human induced pluripotent stem cell (iPSC) lines, iCS-MAF1-1 and iCS-MAF1-11, were generated from fibroblasts. The donor has a heterozygous mutation in the VPS13B gene, which manifests in her child as Cohen syndrome. It is a Golgi pathology, characterized by postnatal microcephaly and delayed growth and mental development. However, the process underlying pathological changes leading to the onset of the disease is still unknown. The use of iPSC will allow describing the early stages of neurogenesis, which is undoubtedly relevant for identifying key stages of development, at which phenotypic manifestations of mutations in the VPS13B gene are found.

Derivation of induced pluripotent stem cells line (RCPCMi007-A-1) with inactivation of the beta-2-microglobulin gene by CRISPR/Cas9 genome editing.

The mismatch of HLA haplotypes between donor and recipient adversely affects the outcome of tissue transplantation. TheB2Mgene knockout (B2M-KO) disrupts the HLA I heterodimer formation; therefore,B2M-KO cells have reduced immunogenicity to allogeneic CD8+ T cells. Thus, theB2M-KO IPSCs and their derivatives can potentially solve a problem of the immunological compatibility in allogeneic transplantations. Using CRISPR/Cas9-mediated genome editing, we generated a human B2M-KO iPSC line (RCPCMi007-A-1). The RCPCMi007-A-1 iPSCs express pluripotency markers, have typical stem cell morphology, maintain normal karyotype, and the ability to differentiate into three germ layers.

Generation of induced pluripotent stem cell line RCPCMi008-A derived from patient with spinocerebellar ataxia 17.

IPSC line RCPCMi004-8 was generated from skin fibroblasts collected from a male patient with spinocerebellar ataxia 17. The patient has expanded trinucleotide CAG repeats in the TBP (TATA-binding protein) gene on chromosome 6q27. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4, and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and the formation of embryoid bodies. The RCPCMi008-A cell line carries the same trinucleotide CAG repeats in the TBP gene. The RCPCMi008-A cell line can be used to model Spinocerebellar ataxia in vitro.

Generation of induced pluripotent stem cell line RCPCMi004-A derived from patient with Parkinson's disease with deletion of the exon 2 in PARK2 gene.

IPSC line RCPCMi004-A was generated from skin fibroblasts collected from a male patient with early onset Parkinson's disease. The patient carries a heterozygous deletion of the exon 2 of PARK2 gene. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4 and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and formation of embryoid bodies. The RCPCMi004-A cell line carries the same deletion in PARK2 gene. The RCPCMi004-A cell line can be used to model Parkinson's disease in vitro.

[The Role of Mutant RNA in the Pathogenesis of Huntington's Disease and Other Polyglutamine Diseases].

Polyglutamine diseases are rare, inherited neurodegenerative pathologies that arise as a result of expansion of trinucleotide CAG repeats in the coding segment of certain genes. This expansion leads to the appearance of mRNA with abnormally long repetitive CAG triplets (mCAG-RNA) and proteins with polyglutamine (PolyQ) tracts in the cells, which is why these pathologies are commonly termed polyglutamine diseases, or PolyQ diseases. To date, nine PolyQ diseases have been described: Huntington's disease, dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six different types of spinocerebellar ataxia (SCA 1,2,3,6,7, and 17). PolyQ diseases lead to serious, constantly progressing dysfunctions of the nervous and/or muscular systems, and there currently exists no efficacious therapy for any of them. Recent studies have convincingly shown that mCAG-RNA can actively participate in the pathological process during the development of PolyQ diseases. Mutant RNA is involved in a wide range of molecular mechanisms, ultimately leading to disruption of the functions of transcription, splicing, translation, cytosol structure, RNA transport from the nucleus to the cytoplasm, and, finally, to neurodegeneration. This review discusses the involvement of mutant mCAG-RNA in neurodegenerative processes in PolyQ diseases.

[At Home among Strangers: Is It Possible to Create Hypoimmunogenic Pluripotent Stem Cell Lines?]

Human pluripotent stem cells, which include embryonic stem cells and induced pluripotent cells (iPSCs), are capable of unlimited division and differentiation into all cells of the body. These cells are considered as a potential source of various types of cells for transplantations. The use of autologous iPSCs is not potentially associated with immune rejection and does not require immunosuppression required for allogeneic grafts. However, the high cost of this technology and the duration of obtaining iPSCs and differentiated cells may limit the use of autologous iPSCs in clinical practice. In addition, full equivalence and immunological compatibility of autologous iPSCs and their derivatives have been repeatedly questioned. One approach to solving the problem of the immunological compatibility of allogeneic derivatives of iPSCs can be the establishment of cell lines with reduced immunogenicity. Differentiated derivatives of such iPSCs may be suitable for transplantation to any patient. This review discusses the strategies for evading immune surveillance in normal and tumor processes that can be used to establish stem cell lines with reduced immunogenicity.

Such Various Stem Cells.

Perhaps there is no more intriguing topic in modern biology than stem cells. The growing interest in stem cells is dictated by the ability of stem cells to both self-renew and differentiate, at least into several type cells. If we learn to influence these properties or reproduce them in vitro, it will be possible to effectively use stem cells or their differentiated derivatives in medicine. Fundamental knowledge of mechanisms of the self-maintenance and differentiation of stem cells is important for understanding a variety of processes - from embryogenesis to aging and oncogenic transformation. The purpose of this issue is to introduce readers to different areas in research on mammalian stem cells, including human stem cells. In the issue both review articles and research papers are presented, and the authors hope that they will be of interest for biochemists, cell biologists, and specialists in the field of biomedicine.

Development of Hematopoietic Stem Cells in the Early Mammalian Embryo.

Hematopoietic stem cells (HSCs) were the first stem cells discovered in humans. A. A. Maximov proposed an idea of blood stem cells that was confirmed later by McCulloch and Till experimentally. HSCs were the first type of stem cells to be used in clinics and ever since are being continually used. Indeed, a single HSC transplanted intravenously is capable of giving rise to all types of blood cells. In recent decades, human and animal HSC origin, development, hierarchy, and gene signature have been extensively investigated. Due to the constant need for donor blood and HSCs suitable for therapeutic transplants, the experimental possibility of obtaining HSCs in vitro by directed differentiation of pluripotent stem cells (PSCs) has been considered in recent years. However, despite all efforts, it is not yet possible to reproduce in vitro the ontogenesis of HSCs and obtain cells capable of long-term maintenance of hematopoiesis. The study of hematopoiesis in embryonic development facilitates the establishment and improvement of protocols for deriving blood cells from PCSs and allows a better understanding of the pathogenesis of various types of proliferative blood diseases, anemia, and immunodeficiency. This review focuses on the development of hematopoiesis in mammalian ontogenesis.

"Necessity Is the Mother of Invention" or Inexpensive, Reliable, and Reproducible Protocol for Generating Organoids.

Organoids are three-dimensional (3D) cell cultures that replicate some of the key features of morphology, spatial architecture, and functions of a particular organ. Organoids can be generated from both adult and pluripotent stem cells (PSCs), and complex organoids can also be obtained by combining different types of cells, including differentiated cells. The ability of pluripotent cells to self-organize into organotypic structures containing several cell subtypes specific for a particular organ was used for creating organoids of the brain, eye, kidney, intestine, and other organs. Despite the advantages of using PSCs for obtaining organoids, an essential shortcoming that prevents their widespread use has been a low yield when they are obtained from a PSC monolayer culture and a large variation in size. This leads to great heterogeneity on further differentiation. In this article, we describe our own protocol for generating standardized organoids, with emphasis on a method for generating brain organoids, which allows scaling-up experiments and makes their cultivation less expensive and easier.

Pluripotent Stem Cells for Modelling and Cell Therapy of Parkinson's Disease.

Studying pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD), requires adequate disease models. The available patient's material is limited to biological fluids and post mortem brain samples. Disease modeling and drug screening can be done in animal models, although this approach has its own limitations, since laboratory animals do not suffer from many neurodegenerative diseases, including PD. The use of neurons obtained by targeted differentiation from induced pluripotent stem cells (iPSCs) with known genetic mutations, as well as from carriers of sporadic forms of the disease, will allow to elucidate new components of the molecular mechanisms of neurodegeneration. Such neuronal cultures can also serve as unique models for testing neuroprotective compounds and monitoring neurodegenerative changes against a background of various therapeutic interventions. In the future, dopaminergic neurons differentiated from iPSCs can be used for cell therapy of PD.

Possibilities for Using Pluripotent Stem Cells for Restoring Damaged Eye Retinal Pigment Epithelium.

The retinal pigment epithelium is a monolayer of pigmented, hexagonal cells connected by tight junctions. These cells compose part of the outer blood-retina barrier, protect the eye from excessive light, have important secretory functions, and support the function of photoreceptors, ensuring the coordination of a variety of regulatory mechanisms. It is the degeneration of the pigment epithelium that is the root cause of many retinal degenerative diseases. The search for reliable cell sources for the transplantation of retinal pigment epithelium is of extreme urgency. Pluripotent stem cells (embryonic stem or induced pluripotent) can be differentiated with high efficiency into the pigment epithelium of the retina, which opens up possibilities for cellular therapy in macular degeneration and can slow down the development of pathology and, perhaps, restore a patient's vision. Pioneering clinical trials on transplantation of retinal pigment epithelial cells differentiated from pluripotent stem cells in the United States and Japan confirmed the need for developing and optimizing such approaches to cell therapy. For effective use, pigment epithelial cells differentiated from pluripotent stem cells should have a set of functional properties characteristic of such cells in vivo. This review summarizes the current state of preclinical and clinical studies in the field of retinal pigment epithelial transplantation therapy. We also discuss different differentiation protocols based on data in the literature and our own data, and the problems holding back the widespread therapeutic application of retinal pigment epithelium differentiated from pluripotent stem cells.

Differentiation of Human Pluripotent Stem Cells into Mesodermal and Ectodermal Derivatives Is Independent of the Type of Isogenic Reprogrammed Somatic Cells.

Induced pluripotent stem cells (iPSCs) have the capacity to unlimitedly proliferate and differentiate into all types of somatic cells. This capacity makes them a valuable source of cells for research and clinical use. However, the type of cells to be reprogrammed, the selection of clones, and the various genetic manipulations during reprogramming may have an impact both on the properties of iPSCs and their differentiated derivatives. To assess this influence, we used isogenic lines of iPSCs obtained by reprogramming of three types of somatic cells differentiated from human embryonic stem cells. We showed that technical manipulations in vitro, such as cell sorting and selection of clones, did not lead to the bottleneck effect, and that isogenic iPSCs derived from different types of somatic cells did not differ in their ability to differentiate into the hematopoietic and neural directions. Thus, the type of somatic cells used for the generation of fully reprogrammed iPSCs is not important for the practical and scientific application of iPSCs.

[Genetic Cell Reprogramming: A New Technology for Basic Research and Applied Usage].

Gene function disclosure and the development of modern technologies of genetic manipulations offered the possibility of genetic reprogramming application to alter cell specialization. With the involvement of a gene set that encodes the transcription factors responsible for the pluripotent state, any cell of an adult body could be reprogrammed into the embryonal.state and pluripotency could be induced in this cell. Such reprogrammed cells were called induced pluripotent stem cells (iPSCs), and they are capable of again passing through all developmental stages. This provides new possibilities for studies of the basic mechanisms of developmental biology, the formation of specific cell types, and the whole body. In culture, iPSCs could be maintained permanently in a nontransformed state and permit genetic manipulations while maintaining their pluripotent properties. Such a unique combination of their properties makes them an attractive tool for studies of various pathologies and for the delineation of treatment approaches. This review discusses the basic and applied aspects of iPSCs biology.

No DNA damage response and negligible genome-wide transcriptional changes in human embryonic stem cells exposed to terahertz radiation.

Terahertz (THz) radiation was proposed recently for use in various applications, including medical imaging and security scanners. However, there are concerns regarding the possible biological effects of non-ionising electromagnetic radiation in the THz range on cells. Human embryonic stem cells (hESCs) are extremely sensitive to environmental stimuli, and we therefore utilised this cell model to investigate the non-thermal effects of THz irradiation. We studied DNA damage and transcriptome responses in hESCs exposed to narrow-band THz radiation (2.3 THz) under strict temperature control. The transcription of approximately 1% of genes was subtly increased following THz irradiation. Functional annotation enrichment analysis of differentially expressed genes revealed 15 functional classes, which were mostly related to mitochondria. Terahertz irradiation did not induce the formation of γH2AX foci or structural chromosomal aberrations in hESCs. We did not observe any effect on the mitotic index or morphology of the hESCs following THz exposure.

Patient-Specific Induced Pluripotent Stem Cells for SOD1-Associated Amyotrophic Lateral Sclerosis Pathogenesis Studies.

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells, called induced pluripotent stem cells (iPSCs), can be an unlimited source of specialized cell types for the body. Thus, autologous somatic cell replacement therapy becomes possible, as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited, and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons.

Molecular barriers to processes of genetic reprogramming and cell transformation.

Genetic reprogramming by ectopic expression of transcription factor genes induces the pluripotent state in somatic cells. This technology provides an opportunity to establish pluripotent stem cells for each person, as well as to get better understanding of epigenetic mechanisms controlling cell state. Interestingly, some of the molecular processes that accompany somatic cell reprogramming in vitro are also characteristic for tumor manifestation. Thus, similar "molecular barriers" that control the stability of epigenetic state exist for both processes of pluripotency induction and malignant transformation. The reprogramming of tumor cells is interesting in two aspects: first, it will determine the contribution of epigenetic changes in carcinogenesis; second, it gives an approach to evaluate tumor stem cells that are supposed to form the entire cell mass of the tumor. This review discusses the key stages of genetic reprogramming, the similarity and difference between the reprogramming process and malignant transformation.