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.

[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.

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.

"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.

[Allocation of precursor germinal cells from the tissue of the human ovaries].

The article present the results of a study on the allocation of precursor germinal cells from tissue of human ovaries obtained during various gynecological operations. Two series of cell culture were isolated from tissue of tunica albuginea. The cell lines were identified and characterized by PCR012-method, immunocytochemical staining and histological examination. The study provided data reliably confirming the presence of precursor cells in the ovarian tissue of women in their reproductive years.

[Comparison of sevoflurane and propofol anesthesia for aortocoronary bypass surgery without artificial circulation].

UNLABELLED: The purpose of the study was to compare the effectiveness of sevoflurane and propofol during combined anesthesia with epidural component during aortocoronary bypass surgery without artificial circulation. MATERIALS AND METHODS: The study included 24 patients with ischemic heart disease. All patients underwent aortocoronary bypass surgery on the working heart (from 1 to 5 bypasses) under combined anesthesia (propofol or sevoflurane with epidural anesthesia with the use of ropicavaine and fentanyl). Induction of anesthesia was carried out by midasolam 0.06 mg/kg, propofol 1-1.5 mg/kg, fentanyl 2.5 mcg/kg, myorelaxation was reached by pipecuronium bromide 0.1 mg/kg. Patients were randomized into propofol group (n = 12) and sevoflurane group (n = 12). Both groups received low flow anesthesia (1l/min) with the use of Drager Primus anesthesia workstation. Anesthesia in the first group was maintained by propofol 3-5 mg/kg/h, in the second group by sevoflurane 0.5-3 vol.%. In both groups additional fentanyl was administered in the dose of 1.2 -1.5 mcg/kg/h. RESULTS: In the postoperative period the increase of the stroke volume and decrease of blood plasma lactate were noticed in the sevoflurane group. The postoperative pain, 6 hours after the aortocoronary bypass surgery in the control group was evidently higher according to Visual Analogue Scale. CONCLUSION: Use of sevorane as a component of combined anesthesia during aortocoronary bypass surgery allows to improve the performance of the myocardium, reduce the severity of hypoperfusion in the perioperative period and reduce the severity of pain after the surgery compared to propofol anesthesia.

Mouse hepatocytes retain the expression of the main differentiation markers during culturing on collagen-chitosan matrices.

Mouse hepatocytes cultured on artificial 3D collagen-chitosan biopolymer matrices retained the expression of hepatocyte markers for 14 days.

Single transpulmonary thermodilution and continuous monitoring of central venous oxygen saturation during off-pump coronary surgery.

BACKGROUND: Off-pump coronary artery bypass grafting (OPCAB) requires thorough monitoring of hemodynamics and oxygen transport. Our aim was to find out whether therapeutic guidance during and after OPCAB, using an algorithm based on advanced monitoring, influences perioperative hemodynamic and fluid management as well as the length of post-operative ICU and hospital stay. METHODS: Patients were randomized into two groups of hemodynamic monitoring: the conventional monitoring (CM) group (n=20) and the advanced monitoring (AM) group (n=20). In the CM group, therapy was guided by central venous pressure, mean arterial pressure (MAP) and heart rate (HR), and in the AM group by the intrathoracic blood volume index, MAP, HR, central venous oxygen saturation (ScvO(2)) and cardiac index (CI). The measurements were performed before and during surgery, and at 2, 4 and 6 h post-operatively. RESULTS: In the AM group, colloids and dobutamine were given more frequently and were accompanied by increments in ScvO(2), CI and oxygen delivery compared with baseline. The percentage of ephedrine administration was higher in the CM group. The algorithm guided by AM decreased time until achieving the status of 'fit for ICU discharge' and post-operative hospital stay by 15% and 25%, respectively. CONCLUSIONS: A goal-directed algorithm based on advanced hemodynamic monitoring and continuous measurement of ScvO(2) facilitates early detection and correction of hemodynamic changes and influences the strategy for fluid therapy that can improve the course of post-operative period after coronary artery bypass grafting on the beating heart.

[Different sensitivity of inbred mice to hepatocarcinogen ortho-aminoazotoluene may be due to differences in the negative control mechanisms of hepatocyte proliferation]. / Raznaia chuvsvitel'nost' myshei inbrednykh linii k gepatokantserogenu orto-aminoazotoluolu mozhet byt' sviazana s razlichiiami v mekhanizmakh negativnogo kontrolia prolifaratsii gepatotsitov.

A most convenient model to study mechanisms of live organism response to chemical carcinogens is tumor induction in murine liver by aminoazodyes, in particular by ortho-aminoazotoluene (OAT). We studied both early and late stages of hepatocarcinogenesis on several lines of inbred mice differing in sensibility to OAT. By means of autoradiography, we examined proliferative activity of hepatocytes obtained from the liver of sensitive (A/He, DD, SWR) and resistant to OAT AKR, CC57Br, BALB/c lines of mice, which were injected carcinogen. The level of p53, p21Cip1, bax, mdm2, cyclin G, gadd45 genes expression in the liver of mice of different lines given OAT injection was studied by multiplex PCR method. Carcinogen caused a decrease of hepatocyte proliferative activity induced by partial hypatectomy (PHE), and an increase in p53, p21Cip, bax, mdm2, and cyclin G genes within mice of A/He, DD and SWR lines. Cell fusion experiments on hepatocytes obtained from regenerating murine liver sensitive to A/He line carcinogen and given long-time OAT administrations with resting and proliferating fibroblasts of NIH 3T3 mice revealed no obvious suppression of DNA synthesis in heterokaryons. Unlike, in fusion experiments on serum-stimulated fibroblasts with hepatocytes obtained from the liver of BALB/c line mice also given OAT suppression of DNA synthesis in stimulated fibroblasts in heterokaryons was observed 15 days following PHE. These results enable us to conclude that OAT administrations break negative endogenous mechanisms of hepatocyte proliferation control in the liver of mice sensitive to carcinogenes.

[Inhibitors of protein biosynthesis can stimulate proliferation of mouse hepatocytes in vitro]. / Ingibitory biosinteza belka mogut stimulirovat' proliferativnuiu aktivnost' gepatotsitov myshi in vitro.

Hepatocyte proliferation in the liver regenerating after partial hepatectomy ceases when the organ is restored, and the mechanism of this phenomenon is still unclear. In the experiments on fusing hepatocytes from the regenerated mouse liver (15 days after partial hepatectomy) with NIH 3T3 mouse fibroblasts, we revealed no DNA synthesis in the nuclei of stimulated fibroblasts in heterokaryons (in the presence of hepatocyte nuclei), whereas DNA synthesis in nonfused cells was undisturbed. In this work, our purpose was to find out whether the suppression of DNA synthesis in heterokaryons could be due to the appearance in hepatocytes of some endogenous factors having an inhibitory effect on proliferation. To this end, hepatocytes from the mouse liver regenerated after partial hepatectomy were treated with cycloheximide for 1-4 h and were then fused with stimulated fibroblasts. Such a short-term treatment of hepatocytes with cycloheximide proved to result in the loss of their ability to inhibit DNA synthesis in the nuclei of stimulated or quiescent fibroblasts in heterokaryons, but hepatocytes proper actively proliferated in the medium with a low serum content (0.2%). When the mice with the liver regenerated after partial hepatectomy were treated with a single sublethal dose of cycloheximide (3 mg/kg), their hepatocytes taken two days after this treatment had no inhibitory effect. Puromycin, another inhibitor o protein synthesis, had the same effect on hepatocytes. These results may be interpreted as evidence that the final stage of liver regeneration after damage is controlled by the factors having a negative effect on cell proliferation.

Dobutamine prevents experimental postintoxication liver cirrhosis in mice.

Various chronic inflammatory and necrotic processes in the liver parenchyma are accompanied by pathological morphofunctional changes, which are associated with hepatocyte death and hyperplasia of the connective tissue. Regeneration of the liver parenchyma should include not only prevention of fibrosis, but also stimulation of hepatocyte proliferation. The adrenoceptor agonist dobutamine stimulated proliferative activity of cultured hepatocytes and prevented the development of postintoxication liver cirrhosis in mice produced by chronic poisoning with CCl(4).

[Hepatocytes in heterokaryons can suppress entry into the S-period of fibroblast nuclei from murine NIH 3T3 cells]. / Gepatotsity v geterokarionakh mogut podavliat' vstuplenie v S-period iader fibroblastov myshi linii NIH 3T3.

Mouse liver regeneration after partial hepatectomy can be considered as a spectacular example of controlled tissue increase. In this study serum-deprived (0.2%) resting and serum-stimulated (10%) proliferating NIH 3T3 mouse fibroblasts were fused with primary hepatocytes isolated from normal (intact) and regenerating adult mouse liver at different times after partial hepatectomy (1-15 days) to elucidate mechanisms of liver cell proliferation cessation at the regeneration end. DNA synthesis was investigated in the nuclei of heterokaryons and non-fused cells using radioautography. Hepatocytes isolated from regenerating liver within 1-12 days following operation did not retard the entry of stimulated fibroblast nuclei into the S-period. In contrast, hepatocytes isolated within 15 days after hepatectomy were found to have inhibitory effect on the entry of stimulated fibroblast nuclei into the S-period in heterokaryons. Preincubation of these hepatocytes with cyclocheximide for 2-4 h abolished their ability to suppress DNA synthesis in stimulated fibroblast nuclei in heterokaryons. Possible reasons of inhibitory effect of differentiated cells in heterokaryos are discussed. The data obtained enable us to conclude that the mechanism of proliferative process control in regenerating hepatocytes seems to be stopped being affected by the intracellular growth inhibitors, whose formation depends on protein synthesis.