Single-cell atlas of healthy human blood unveils age-related loss of NKG2C+GZMB-CD8+ memory T cells and accumulation of type 2 memory T cells.

Extensive, large-scale single-cell profiling of healthy human blood at different ages is one of the critical pending tasks required to establish a framework for the systematic understanding of human aging. Here, using single-cell RNA/T cell receptor (TCR)/BCR-seq with protein feature barcoding, we profiled 317 samples from 166 healthy individuals aged 25-85 years old. From this, we generated a dataset from ∼2 million cells that described 55 subpopulations of blood immune cells. Twelve subpopulations changed with age, including the accumulation of GZMK+CD8+ T cells and HLA-DR+CD4+ T cells. In contrast to other T cell memory subsets, transcriptionally distinct NKG2C+GZMB-CD8+ T cells counterintuitively decreased with age. Furthermore, we found a concerted age-associated increase in type 2/interleukin (IL)4-expressing memory subpopulations across CD4+ and CD8+ T cell compartments (CCR4+CD8+ Tcm and Th2 CD4+ Tmem), suggesting a systematic functional shift in immune homeostasis with age. Our work provides novel insights into healthy human aging and a comprehensive annotated resource.

The Effects of Immunosuppressive Drugs on the Characteristics and Functional Properties of Bone Marrow-Derived Stem Cells Isolated from Patients with Diabetes Mellitus and Peripheral Arterial Disease.

BACKGROUND: Diabetic patients (DPs) with foot ulcers can receive autologous cell therapy (ACT) as a last therapeutic option. Even DPs who have undergone organ transplantation and are using immunosuppressive (IS) drugs can be treated by ACT. The aim of our study was to analyze the effects of IS drugs on the characteristics of bone marrow-derived stem cells (BM-MSCs). METHODS: The cells were isolated from the bone marrow of DPs, cultivated for 14-18 days, and phenotypically characterized using flow cytometry. These precursor cells were cultured in the presence of various IS drugs. The impact of IS drugs on metabolic activity was measured using a WST-1 assay, and the expression of genes for immunoregulatory molecules was detected through RT-PCR. Cell death was analyzed through the use of flow cytometry, and the production of cytokines was determined by ELISA. RESULTS: The mononuclear fraction of cultured cells contained mesenchymal stem cells (CD45-CD73+CD90+CD105+), myeloid angiogenic cells (CD45+CD146-), and endothelial colony-forming cells (CD45-CD146+). IS drugs inhibited metabolic activity, the expression of genes for immunoregulatory molecules, the production of cytokines, and the viability of the cells. CONCLUSIONS: The results indicate that IS drugs in a dose-dependent manner had a negative impact on the properties of BM-MSCs used to treat ischemic diabetic foot ulcers, and that these drugs could affect the therapeutic potential of BM-MSCs.

Metal Nanoparticles with Antimicrobial Properties: The Toxicity Response in Mouse Mesenchymal Stem Cells.

Some metal nanoparticles (NP) are characterized by antimicrobial properties with the potential to be used as alternative antibiotics. However, NP may negatively impact human organism, including mesenchymal stem cells (MSC), a cell population contributing to tissue growth and regeneration. To address these issues, we investigated the toxic effects of selected NP (Ag, ZnO, and CuO) in mouse MSC. MSC were treated with various doses of NP for 4 h, 24 h, and 48 h and multiple endpoints were analyzed. Reactive oxygen species were generated after 48 h CuO NP exposure. Lipid peroxidation was induced after 4 h and 24 h treatment, regardless of NP and/or tested dose. DNA fragmentation and oxidation induced by Ag NP showed dose responses for all the periods. For other NP, the effects were observed for shorter exposure times. The impact on the frequency of micronuclei was weak. All the tested NP increased the sensitivity of MSC to apoptosis. The cell cycle was most affected after 24 h, particularly for Ag NP treatment. In summary, the tested NP induced numerous adverse changes in MSC. These results should be taken into consideration when planning the use of NP in medical applications where MSC are involved.

The Impact of Metal Nanoparticles on the Immunoregulatory and Therapeutic Properties of Mesenchymal Stem Cells.

Negative impacts of nanomaterials on stem cells and cells of the immune system are one of the main causes of an impaired or slowed tissue healing. Therefore, we tested effects of four selected types of metal nanoparticles (NPs): zinc oxide (ZnO), copper oxide (CuO), silver (Ag), and titanium dioxide (TiO2) on the metabolic activity and secretory potential of mouse mesenchymal stem cells (MSCs), and on the ability of MSCs to stimulate production of cytokines and growth factors by macrophages. Individual types of nanoparticles differed in the ability to inhibit metabolic activity, and significantly decreased the production of cytokines and growth factors (interleukin-6, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor-1) by MSCs, with the strongest inhibitory effect of CuO NPs and the least effect of TiO2 NPs. The recent studies indicate that immunomodulatory and therapeutic effects of transplanted MSCs are mediated by macrophages engulfing apoptotic MSCs. We co-cultivated macrophages with heat-inactivated MSCs which were untreated or were preincubated with the highest nontoxic concentrations of metal NPs, and the secretory activity of macrophages was determined. Macrophages cultivated in the presence of both untreated MSCs or MSCs preincubated with NPs produced significantly enhanced and comparable levels of various cytokines and growth factors. These results suggest that metal nanoparticles inhibit therapeutic properties of MSCs by a direct negative effect on their secretory activity, but MSCs cultivated in the presence of metal NPs have preserved the ability to stimulate cytokine and growth factor production by macrophages.

Effects of antimicrobial metal nanoparticles on characteristics and function properties of mouse mesenchymal stem cells.

Nanoparticles (NPs) have a wide use in various field of industry and in medicine, where they represent a promise for their antimicrobial effects. Simultaneous application of NPs and therapeutic stem cells can speed up tissue regeneration and improve healing process but there is a danger of negative impacts of NPs on stem cells. Therefore, we tested effects of four types of metal antimicrobial NPs on characteristics and function properties of mouse mesenchymal stem cells (MSCs) in vitro. All types of tested NPs, i.e. zinc oxide, silver, copper oxide and titanium dioxide, exerted negative effects on the expression of phenotypic markers, metabolic activity, differentiation potential, expression of genes for immunoregulatory molecules and on production of cytokines and growth factors by MSCs. However, there were apparent differences in the impact of individual types of NPs on tested characteristics and function properties of MSCs. The results showed that individual types of NPs influence the activity of MSCs, and thus the use of metal NPs during tissue regeneration and in combination with stem cell therapy should be well considered.

The Inability of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells to Survive in Newborn Mice and to Induce Transplantation Tolerance.

An encounter of the developing immune system with an antigen results in the induction of immunological areactivity to this antigen. In the case of transplantation antigens, the application of allogeneic hematopoietic cells induces a state of neonatal transplantation tolerance. This tolerance depends on the establishment of cellular chimerism, when allogeneic cells survive in the neonatally treated recipient. Since mesenchymal stem/stromal cells (MSCs) have been shown to have low immunogenicity and often survive in allogeneic recipients, we attempted to use these cells for induction of transplantation tolerance. Newborn (less than 24 h old) C57BL/6 mice were injected intraperitoneally with 5 × 106 adipose tissue-derived MSCs isolated from allogeneic donors and the fate and survival of these cells were monitored. The impact of MSC application on the proportion of cell populations of the immune system and immunological reactivity was assessed. In addition, the survival of skin allografts in neonatally treated recipients was tested. We found that in vitro expanded MSCs did not survive in neonatal recipients, and the living MSCs were not detected few days after their application. Furthermore, there were no significant changes in the proportion of individual immune cell populations including CD4+ cell lineages, but we detected an apparent shift to the production of Th1 cytokines IL-2 and IFN-γ in neonatally treated mice. However, skin allografts in the MSC-treated recipients were promptly rejected. These results therefore show that in vitro expanded MSCs do not survive in neonatal recipients, but induce a cytokine imbalance without induction of transplantation tolerance.

The Altered Migration and Distribution of Systemically Administered Mesenchymal Stem Cells in Morphine-Treated Recipients.

Mesenchymal stem cells (MSCs) have the ability to migrate to the site of injury or inflammation, and to contribute to the healing process. Since patients treated with MSCs are often users of analgesic drugs, to relieve their uncomfortable pain associated with the tissue disorder, there is a possibility of negative effects of these drugs on the migration of endogenous and exogenous MSCs. Therefore, we tested the impact of acute and chronic treatment with morphine on the migration and organ distribution of exogenous adipose tissue-derived MSCs in mouse models. Firstly, we showed that the incubation of MSCs with morphine significantly reduced the expression of adhesive molecules CD44 (HCAM), CD54 (ICAM-1) and CD106 (VCAM-1) on MSCs. Using a model of systemic administration of MSCs labeled with vital dye PKH26 and by the application of flow cytometry to detect living CD45-PKH26+ cells, we found a decreased number of labeled MSCs in the lung, spleen and bone marrow, and a significantly increased number of MSCs in the liver of morphine-treated recipients. A skin allograft model was used to study the effects of morphine on the migration of exogenous MSCs to the superficial wound. Intraperitoneally administered MSCs migrated preferentially to the wound site, and this migration was significantly decreased in the morphine-treated recipients. The present results showed that morphine significantly influences the distribution of exogenous MSCs in the body, and decreases their migration to the site of injury.