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Background: Standard approaches to the treatment of chronic post-radiation proctitis are associated with a high risk of complications and a high percentage of unsatisfactory results due to the reduced regenerative potential of irradiated tissues. Regenerative surgery techniques using the stromal-vascular cell fraction (SVF) based on the patient's autologous adipose tissue are a promising direction for study., Clinical Case Description: A 76-year-old patient suffering from chronic post-radiation erosive-ulcerative proctitis, grade 4 according to RTOG-EORTC, complicated by recurrent profuse rectal bleeding, underwent local autotransplantation of SVF into the submucosal layer of the rectum and pararectal connective tissue. The follow-up colonoscopies 1 and 6 months after the surgery and histological examination showed the complete epithelialization of ulcerative defects and a decrease in proctitis activity. There were no bleeding episodes during the 12-month postoperative observation period., Conclusion: The proangiogenic, wound-healing, and anti-apoptotic effects of the SVF cell suspension provided reduction of inflammation activity, epithelialization of ulcers, and elimination of defecation-associated hemorrhage, following the SVF injection into the submucosal layer of the rectal wall and pararectal connective tissue in a patient with post-radiation proctitis with ulcers and recurrent bleeding., (© 2024. The Author(s).)

Hepatitis C virus (HCV) is one of the basic culprits behind chronic liver disease, which may result in cirrhosis and hepatocarcinoma. In spite of the extensive research conducted, a vaccine against HCV has not been yet created. We have obtained human mesenchymal stem cells (hMSCs) and used them for expressing the HCV NS5A protein as a model vaccination platform. Sixteen hMSC lines of a different origin were transfected with the pcNS5A-GFP plasmid to obtain genetically modified MSCs (mMSCs). The highest efficiency was obtained by the transfection of dental pulp MSCs. C57BL/6 mice were immunized intravenously with mMSCs, and the immune response was compared with the response to the pcNS5A-GFP plasmid, which was injected intramuscularly. It was shown that the antigen-specific lymphocyte proliferation and the number of IFN-γ-synthesizing cells were two to three times higher after the mMSC immunization compared to the DNA immunization. In addition, mMSCs induced more CD4+ memory T cells and an increase in the CD4+/CD8+ ratio. The results suggest that the immunostimulatory effect of mMSCs is associated with the switch of MSCs to the pro-inflammatory phenotype and a decrease in the proportion of myeloid derived suppressor cells. Thus, the possibility of using human mMSCs for the creation of a vaccine against HCV has been shown for the first time.

We developed recombinant variants of oncolytic vaccinia virus LIVP strain expressing interleukin-15 (IL-15) or its receptor subunit alpha (IL-15Rα) to stimulate IL-15-dependent immune cells. We evaluated their oncolytic activity either alone or in combination with each other in vitro and in vivo using the murine CT26 colon carcinoma and 4T1 breast carcinoma models. We demonstrated that the admixture of these recombinant variants could promote the generation of the IL-15/IL-15Rα complex. In vitro studies indicated that 4T1 breast cancer cells were more susceptible to the developed recombinant viruses. In vivo studies showed significant survival benefits and tumor regression in 4T1 breast cancer syngeneic mice that received a combination of LIVP-IL15-RFP with LIVP-IL15Ra-RFP. Histological analysis showed recruited lymphocytes at the tumor region, while no harmful effects to the liver or spleen of the animals were detected. Evaluating tumor-infiltrated lymphocytes represented profound activation of cytotoxic T cells and macrophages in mice receiving combination therapy. Thus, our experiments showed superior oncolytic effectiveness of simultaneous injection of LIVP-IL15-RFP and LIVP-IL15Ra-RFP in breast cancer-bearing mice. The combined therapy by these recombinant variants represents a potent and versatile approach for developing new immunotherapies for breast cancer., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 The Authors.)

For the first time, the data have been obtained on the effects of high-intensity terahertz (THz) radiation (with the intensity of 30 GW/cm 2 , electric field strength of 3.5 MV/cm) on human skin fibroblasts. A quantitative estimation of the number of histone Н2АХ foci of phosphorylation was performed. The number of foci per cell was studied depending on the irradiation time, as well as on the THz pulse energy. The performed studies have shown that the appearance of the foci is not related to either the oxidative stress (the cells preserve their morphology, cytoskeleton structure, and the reactive oxygen species content does not exceed the control values), or the thermal effect of THz radiation. The prolonged irradiation of fibroblasts also did not result in a decrease of their proliferative index., Competing Interests: The authors declare no conflicts of interest., (© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

Exposure of cells or biological tissues to high-power pulses of terahertz (THz) radiation leads to changes in a variety of intracellular processes. However, the role of heating effects due to strong absorption of THz radiation by water molecules still stays unclear. In this study, we performed numerical modelling in order to estimate the thermal impact on water of a single THz pulse as well as a series of THz pulses. A finite-element (FE) model that provides numerical solutions for the heat conduction equation is employed to compute the temperature increase. A simple expression for temperature estimation in the center of the spot of THz radiation is presented for given frequency and fluence of the THz pulse. It has been demonstrated that thermal effect is determined by either the average power of radiation or by the fluence of a single THz pulse depending on pulse repetition rate. Human dermal fibroblasts have been exposed to THz pulses (with an energy of [Formula: see text] and repetition rate of 100 Hz) to estimate the thermal effect. Analysis of heat shock proteins expression has demonstrated no statistically significant difference ([Formula: see text]) between control and experimental groups after 3 h of irradiation., (© 2021. The Author(s).)

Immunotherapy based on adoptive transfer of genetically engineered T- and NK-cells is an area of active ongoing research and has proven highly efficacious for patients with certain B-cell malignancies. Use of NK cells and NK cell lines as carriers of chimeric antigen receptors (CARs) appears particularly promising, as this opens an opportunity for moving the therapy from autologous to the allogeneic (universal) format. This "off-the-shelf" approach is thought to significantly reduce the price of the treatment and make it available to many more patients in need. Yet, the efficacy of CAR-NK cells in vivo presently remains low, and boosting the activity of CAR NK cells via stronger tumor homing, resistance to tumor microenvironment, as well as greater cytotoxicity may translate into improved patient outcomes. Here, we established a derivative of a human NK cell line YT overexpressing a positive regulator of cytotoxicity, VAV1. Activity of YT-VAV1 cells obtained was assayed in vitro against several cancer cell lines and primary patient-derived cancer cells. YT-VAV1 cells outperform parental YT cells in terms of cytotoxicity., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

The interaction of neural progenitor cells (NPCs) with the extracellular matrix (ECM) plays an important role in neural tissue regeneration. Understanding which motifs of the ECM proteins are crucial for normal NPC adhesion, proliferation, and differentiation is important in order to create more adequate tissue engineered models of neural tissue and to efficiently study the central nervous system regeneration mechanisms. We have shown earlier that anisotropic matrices prepared from a mixture of recombinant dragline silk proteins, such as spidroin 1 and spidroin 2, by electrospinning are biocompatible with NPCs and provide good proliferation and oriented growth of neurites. This study objective was to find the effects of spidroin-based electrospun materials, modified with peptide motifs of the extracellular matrix proteins (RGD, IKVAV, and VAEIDGIEL) on adhesion, proliferation, and differentiation of directly reprogrammed neural precursor cells (drNPCs). The structural and biomechanical studies have shown that spidroin-based electrospun mats (SBEM), modified with ECM peptides, are characterized by a uniaxial orientation and elastic moduli in the swollen state, comparable to those of the dura mater. It has been found for the first time that drNPCs on SBEM mostly preserve their stemness in the growth medium and even in the differentiation medium with brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, while addition of the mentioned ECM-peptide motifs may shift the balance toward neuroglial differentiation. We have demonstrated that the RGD motif promotes formation of a lower number of neurons with longer neurites, while the IKVAV motif is characterized by formation of a greater number of NF200-positive neurons with shorter neurites. At the same time, all the studied matrices preserve up to 30% of neuroglial progenitor cells, phenotypically similar to radial glia derived from the subventricular zone. We believe that, by using this approach and modifying spidroin by various ECM-motifs or other substances, one may create an in vitro model for the neuroglial stem cell niche with the potential control of their differentiation., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Background: The development of regenerative therapy for human spinal cord injury (SCI) is dramatically restricted by two main challenges: the need for a safe source of functionally active and reproducible neural stem cells and the need of adequate animal models for preclinical testing. Direct reprogramming of somatic cells into neuronal and glial precursors might be a promising solution to the first challenge. The use of non-human primates for preclinical studies exploring new treatment paradigms in SCI results in data with more translational relevance to human SCI., Aim: To investigate the safety and efficacy of intraspinal transplantation of directly reprogrammed neural precursor cells (drNPCs)., Methods: Seven non-human primates with verified complete thoracic SCI were divided into two groups: drNPC group ( n = 4) was subjected to intraspinal transplantation of 5 million drNPCs rostral and caudal to the lesion site 2 wk post injury, and lesion control ( n = 3) was injected identically with the equivalent volume of vehicle., Results: Follow-up for 12 wk revealed that animals in the drNPC group demonstrated a significant recovery of the paralyzed hindlimb as well as recovery of somatosensory evoked potential and motor evoked potential of injured pathways. Magnetic resonance diffusion tensor imaging data confirmed the intraspinal transplantation of drNPCs did not adversely affect the morphology of the central nervous system or cerebrospinal fluid circulation. Subsequent immunohistochemical analysis showed that drNPCs maintained SOX2 expression characteristic of multipotency in the transplanted spinal cord for at least 12 wk, migrating to areas of axon growth cones., Conclusion: Our data demonstrated that drNPC transplantation was safe and contributed to improvement of spinal cord function after acute SCI, based on neurological status assessment and neurophysiological recovery within 12 wk after transplantation. The functional improvement described was not associated with neuronal differentiation of the allogeneic drNPCs. Instead, directed drNPCs migration to the areas of active growth cone formation may provide exosome and paracrine trophic support, thereby further supporting the regeneration processes., Competing Interests: Conflict-of-interest statement: Authors declare no conflict of interests., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

We evaluated the applicability of chitosan- g -oligo(L,L-lactide) copolymer (CLC) hydrogel for central nervous system tissue engineering. The biomechanical properties of the CLC hydrogel were characterized and its biocompatibility was assessed with neural progenitor cells obtained from two different sources: H9-derived neural stem cells (H9D-NSCs) and directly reprogrammed neural precursor cells (drNPCs). Our study found that the optically transparent CLC hydrogel possessed biomechanical characteristics suitable for culturing human neural stem/precursor cells and was noncytotoxic. When seeded on films prepared from CLC copolymer hydrogel, both H9D-NSC and drNPC adhered well, expanded and exhibited signs of spontaneous differentiation. While H9D-NSC mainly preserved multipotency as shown by a high proportion of Nestin+ and Sox2+ cells and a comparatively lower expression of the neuronal markers βIII-tubulin and MAP2, drNPCs, obtained by direct reprogramming, differentiated more extensively along the neuronal lineage. Our study indicates that the CLC hydrogel may be considered as a substrate for tissue-engineered constructs, applicable for therapy of neurodegenerative diseases. Impact statement We synthetized a chitosan- g -oligo(L,L-lactide) hydrogel that sustained multipotency of embryonic-derived neural stem cells (NSCs) and supported differentiation of directly reprogrammed NSC predominantly along the neuronal lineage. The hydrogel exhibited no cytotoxicity in vitro , both in extraction and contact cytotoxicity tests. When seeded on the hydrogel, both types of NSCs adhered well, expanded, and exhibited signs of spontaneous differentiation. The biomechanical properties of the hydrogel were similar to that of human spinal cord with incised pia mater. These data pave the way for further investigations of the hydrogel toward its applicability in central nervous system tissue engineering.

In in vitro experiments on cultures of human multipotent stem cells from the human bonearrow and dental pulp, we studied direct reprogramming towards neuro-glial lineage cells using a cocktail of small molecules. Reprogramming by the previously published protocol (with a cocktail containing β-mercaptoethanol, LIF, VPA, CHIR99021, and RepSox) and by the optimized protocol (VPA, RG108, А83-01, dorsomorphin, thiazovivin, CHIR99021, forskolin, and Isx9) allows obtaining cells with immunophenotypic and genetic signs of neural stem cells. However, neither the former, nor the optimized protocols allowed preparing neural progenitors capable of adequate terminal differentiation from both bone marrow-derived mesenchymal stem cells and nestin-positive neural crest-derived mesenchymal stem cells. Real-time PCR demonstrated the expression of some neurogenesis markers, but neural stem cell-specific expression pattern was not observed. The findings lead us to a conclusion that reprogramming with small molecules without additional factors modifying gene expression does not allow reproducible production of human neural stem cell-like progenitors that can be used as the source of neural tissue for the regenerative therapy.

We have designed a novel two-component matrix (SPRPix) for the encapsulation of directly reprogrammed human neural precursor cells (drNPC). The matrix is comprised of 1) a solid anisotropic complex scaffold prepared by electrospinning a mixture of recombinant analogues of the spider dragline silk proteins - spidroin 1 (rS1/9) and spidroin 2 (rS2/12) - and polycaprolactone (PCL) (rSS-PCL), and 2) a "liquid matrix" based on platelet-rich plasma (PRP). The combination of PRP and spidroin promoted drNPC proliferation with the formation of neural tissue organoids and dramatically activated neurogenesis. Differentiation of drNPCs generated large numbers of βIII-tubulin and MAP2 positive neurons as well as some GFAP-positive astrocytes, which likely had a neuronal supporting function. Interestingly the SPRPix microfibrils appeared to provide strong guidance cues as the differentiating neurons oriented their processes parallel to them. Implantation of the SPRPix matrix containing human drNPC into the brain and spinal cord of two healthy Rhesus macaque monkeys showed good biocompatibility: no astroglial and microglial reaction was present around the implanted construct. Importantly, the human drNPCs survived for the 3 month study period and differentiated into MAP2 positive neurons. Tissue engineered constructs based on SPRPix exhibits important attributes that warrant further examination in spinal cord injury treatment.

Over many decades, constructing genetically and phenotypically stable lines of neural stem cells (NSC) for clinical purposes with the aim of restoring irreversibly lost functions of nervous tissue has been one of the major goals for multiple research groups. The unique ability of stem cells to maintain their own pluripotent state even in the adult body has made them into the choice object of study. With the development of the technology for induced pluripotent stem cells (iPSCs) and direct transdifferentiation of somatic cells into the desired cell type, the initial research approaches based on the use of allogeneic NSCs from embryonic or fetal nervous tissue are gradually becoming a thing of the past. This review deals with basic molecular mechanisms for maintaining the pluripotent state of embryonic/induced stem and reprogrammed somatic cells, as well as with currently existing reprogramming strategies. The focus is on performing direct reprogramming while bypassing the stage of iPSCs which is known for genetic instability and an increased risk of tumorigenesis. A detailed description of various protocols for obtaining reprogrammed neural cells used in the therapy of the nervous system pathology is also provided.