Dynamic changes in radiological parameters, immune cells, selected miRNAs, and cytokine levels in peripheral blood of patients with severe COVID­19.

The present study aimed to investigate the dynamic changes in peripheral blood leucocyte subpopulations, cytokine and miRNA levels, and changes in computed tomography (CT) scores in patients with severe coronavirus disease 2019 (COVID-19) (n=14) and age-matched non-COVID-19 volunteers (n=17), which were included as a reference control group. All data were collected on the day of patient admission (day 0) and on the 7th, 14th and 28th days of follow-up while CT of the lungs was performed on weeks 2, 8, 24 and 48. On day 0, lymphopenia and leucopenia were detected in most patients with COVID-19, as well as an increase in the percentage of banded neutrophils, B cells, and CD4+ Treg cells, and a decrease in the content of PD-1low T cells, classical, plasmacytoid, and regulatory dendritic cells. On day 7, the percentage of T and natural killer cells decreased with a concurrent increase in B cells, but returned to the initial level after treatment discharge. The content of different T and dendritic cell subsets among CD45+ cells increased during two weeks and remained elevated, suggesting the activation of an adaptive immune response. The increase of PD-1-positive subpopulations of T and non-T cells and regulatory CD4 T cells in patients with COVID-19 during the observation period suggests the development of an inflammation control mechanism. The levels of interferon γ-induced protein 10 (IP-10), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 decreased on day 7, but increased again on days 14 and 28. C-reactive protein and granulocyte colony-stimulating factor (G-CSF) levels decreased gradually throughout the observation period. The relative expression levels of microRNA (miR)-21-5p, miR-221-3p, miR-27a-3p, miR-146a-5p, miR-133a-3p, and miR-126-3p were significantly higher at the beginning of hospitalization compared to non-COVID-19 volunteers. The plasma levels of all miRs, except for miR-126-3p, normalized within one week of treatment. At week 48, CT scores were most prominently correlated with the content of lymphocytes, senescent memory T cells, CD127+ T cells and CD57+ T cells, and increased concentrations of G-CSF, IP-10, and macrophage inflammatory protein-1α.

Age-related ultrastructural changes in spheroids of the adipose-derived multipotent mesenchymal stromal cells from ovariectomized mice.

Introduction: Adipose-derived multipotent mesenchymal stromal cells (ADSCs) are widely used for cell therapy, in particular for the treatment of diseases of the nervous system. An important issue is to predict the effectiveness and safety of such cell transplants, considering disorders of adipose tissue under age-related dysfunction of sex hormones production. The study aimed to investigate the ultrastructural characteristics of 3D spheroids formed by ADSCs of ovariectomized mice of different ages compared to age-matched controls. Methods: ADSCs were obtained from female CBA/Ca mice randomly divided into four groups: CtrlY-control young (2 months) mice, CtrlO-control old (14 months) mice, OVxY-ovariectomized young mice, and OVxO-ovariectomized old mice of the same age. 3D spheroids were formed by micromass technique for 12-14 days and their ultrastructural characteristics were estimated by transmission electron microscopy. Results and Discussion: The electron microscopy analysis of spheroids from CtrlY animals revealed that ADSCs formed a culture of more or less homogeneous in size multicellular structures. The cytoplasm of these ADSCs had a granular appearance due to being rich in free ribosomes and polysomes, indicating active protein synthesis. Extended electron-dense mitochondria with a regular cristae structure and a predominant condensed matrix were observed in ADSCs from CtrlY group, which could indicate high respiratory activity. At the same time, ADSCs from CtrlO group formed a culture of heterogeneous in size spheroids. In ADSCs from CtrlO group, the mitochondrial population was heterogeneous, a significant part was represented by more round structures. This may indicate an increase in mitochondrial fission and/or an impairment of the fusion. Significantly fewer polysomes were observed in the cytoplasm of ADSCs from CtrlO group, indicating low protein synthetic activity. The cytoplasm of ADSCs in spheroids from old mice had significantly increased amounts of lipid droplets compared to cells obtained from young animals. Also, an increase in the number of lipid droplets in the cytoplasm of ADSCs was observed in both the group of young and old ovariectomized mice compared with control animals of the same age. Together, our data indicate the negative impact of aging on the ultrastructural characteristics of 3D spheroids formed by ADSCs. Our findings are particularly promising in the context of potential therapeutic applications of ADSCs for the treatment of diseases of the nervous system.

Stem Cell Therapy Enhances Motor Activity of Triceps Surae Muscle in Mice with Hereditary Peripheral Neuropathy.

Impaired motor and sensory functions are the main features of Charcot-Marie-Tooth disease. Mesenchymal stem cell (MSCs) therapy is one of the possible treatments for this disease. It was assumed that MSCs therapy can improve the contractile properties of the triceps surae (TS) muscles in mice with hereditary peripheral neuropathy. Murine adipose-derived mesenchymal stromal cells (AD-MSCs) were obtained for transplantation into TS muscles of FVB-C-Tg(GFPU)5Nagy/J mice. Three months after AD-MSCs transplantation, animals were subjected to electrophysiological investigations. Parameters of TS muscle tension after intermittent high frequency electrical sciatic nerve stimulations were analyzed. It was found that force of TS muscle tension contraction in animals after AD-MSCs treatment was two-time higher than in untreated mice. Normalized values of force muscle contraction in different phases of electrical stimulation were 0.3 ± 0.01 vs. 0.18 ± 0.01 and 0.26 ± 0.03 vs. 0.13 ± 0.03 for treated and untreated animals, respectively. It is assumed that the two-fold increase in TS muscle strength was caused by stem cell therapy. Apparently, AD-MSCs therapy can promote nerve regeneration and partial restoration of muscle function, and thus can be a potential therapeutic agent for the treatment of peripheral neuropathies.

Adipose-Derived Stem Cells Reduce Lipopolysaccharide-Induced Myelin Degradation and Neuroinflammatory Responses of Glial Cells in Mice.

Brain inflammation is a key event triggering the pathological process associated with many neurodegenerative diseases. Current personalized medicine and translational research in neurodegenerative diseases focus on adipose-derived stem cells (ASCs), because they are patient-specific, thereby reducing the risk of immune rejection. ASCs have been shown to exert a therapeutic effect following transplantation in animal models of neuroinflammation. However, the mechanisms by which transplanted ASCs promote cell survival and/or functional recovery are not fully understood. We investigated the effects of ASCs in in vivo and in vitro lipopolysaccharide (LPS)-induced neuroinflammatory models. Brain damage was evaluated immunohistochemically using specific antibody markers of microglia, astroglia and oligodendrocytes. ASCs were used for intracerebral transplantation, as well as for non-contact co-culture with brain slices. In both in vivo and in vitro models, we found that LPS caused micro- and astroglial activation and oligodendrocyte degradation, whereas the presence of ASCs significantly reduced the damaging effects. It should be noted that the observed ASCs protection in a non-contact co-culture suggested that this effect was due to humoral factors via ASC-released biomodulatory molecules. However, further clinical studies are required to establish the therapeutic mechanisms of ASCs, and optimize their use as a part of a personalized medicine strategy.

Mesenchymal Stem Cells or Interleukin-6 Improve Episodic Memory of Mice Lacking α7 Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) are involved in regulating cognition, inflammation and cell survival. Neuroinflammation is accompanied by the decrease of α7 nAChRs in the brain and impairment of memory. We show here that α7-/- mice possess pro-inflammatory phenotype and demonstrate worse episodic memory compared to wild-type mice. Previously we reported that mesenchymal stem cells (MSCs) restored episodic memory of lipopolysaccharide-treated wild-type mice. The aim of this study was to examine if MSCs or their soluble factors improve memory of α7-/- mice. The α7-specific signal (ELISA) and α7+ cells (IHC) were found in the brain of α7-/- mice on days 7 and 14 after intravenous injection of α7+ MSCs from either human umbilical cord (hMSCs) or mouse placenta (mMSCs). The intravenously injected MSCs or intraperitoneally injected hMSCs-conditioned medium transiently improved episodic memory of α7-/- mice and decreased cytochrome c release from their brain mitochondria under the effect of Ca2+. Either MSCs or conditioned medium stimulated an IL-6 increase in the brain, which coincided with the improvement of episodic memory. Injections of recombinant IL-6 also improved episodic memory of α7-/- mice accompanied by the up-regulation of α3, α4, ß2 and ß4 nAChR subunits in the brain. It is concluded that MSCs, injected intravenously, penetrate the brain of α7-/- mice and persist there for at least 2 weeks. They improve episodic memory of mice and make their mitochondria more resistant to apoptogenic influence. One of the soluble factors responsible for the memory improvement is IL-6.

Intravenously Injected Mesenchymal Stem Cells Penetrate the Brain and Treat Inflammation-Induced Brain Damage and Memory Impairment in Mice.

Neuroinflammation is regarded as one of the pathogenic factors of Alzheimer disease (AD). Previously, we showed that mice regularly injected with bacterial lipopolysaccharide (LPS) possessed the AD-like symptoms like episodic memory decline, elevated amounts of amyloid beta (Aß) peptide (1-42), and decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain. The use of mesenchymal stem cells (MSCs), which can differentiate into multiple cell types, including neurons, is an attractive idea of regenerative medicine, in particular, for neurodegenerative disorders like AD. In the present study, we aimed to investigate whether pathogenic effect of LPS on the brain and behavior of mice can be prevented or treated by injection of MSCs or MSC-produced soluble factors. Fluorescently-labeled MSCs, injected intravenously, were found in the brain blood vessels of LPS-treated mice. Mice co-injected with LPS and MSCs did not demonstrate episodic memory impairment, Aß (1-42) accumulation, and nAChR decrease in the brain and brain mitochondria. Their mitochondria released less cytochrome c under the effect of Ca2+ compared to mitochondria of LPS-only-treated mice. Moreover, MSCs could reverse the pathogenic symptoms developed 3 weeks after LPS injection. Cultured MSCs produced IL-6 in response to LPS and MSCs effect in vivo was accompanied by additional stimulation of both micro- and macroglia. Xenogeneic (human) MSCs were almost as efficient as allogeneic (mouse) ones and regular injections of human MSC-conditioned medium also produced positive effect. These data allow suggesting MSCs as a potential therapeutic tool to cure neuroinflammation-related cognitive pathology.

Maturation of neural stem cells and integration into hippocampal circuits - a functional study in an in situ model of cerebral ischemia.

The hippocampus is the region of the brain that is most susceptible to ischemic lesion because it contains pyramidal neurons that are highly vulnerable to ischemic cell death. A restricted brain neurogenesis limits the possibility of reversing massive cell death after stroke and, hence, endorses cell-based therapies for neuronal replacement strategies following cerebral ischemia. Neurons differentiated from neural stem/progenitor cells (NSPCs) can mature and integrate into host circuitry, improving recovery after stroke. However, how the host environment regulates the NSPC behavior in post-ischemic tissue remains unknown. Here, we studied functional maturation of NSPCs in control and post-ischemic hippocampal tissue after modelling cerebral ischemia in situ We traced the maturation of electrophysiological properties and integration of the NSPC-derived neurons into the host circuits, with these cells developing appropriate activity 3 weeks or less after engraftment. In the tissue subjected to ischemia, the NSPC-derived neurons exhibited functional deficits, and differentiation of embryonic NSPCs to glial types - oligodendrocytes and astrocytes - was boosted. Our findings of the delayed neuronal maturation in post-ischemic conditions, while the NSPC differentiation was promoted towards glial cell types, provide new insights that could be applicable to stem cell therapy replacement strategies used after cerebral ischemia.