Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated β-galactosidase activity (SA-βgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-βgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies

Immune Repertoire Profiling Reveals that Clonally Expanded B and T Cells Infiltrating Diseased Human Kidneys Can Also Be Tracked in Blood

<div><p>Recent advances in high-throughput sequencing allow for the competitive analysis of the human B and T cell immune repertoire. In this study we compared Immunoglobulin and T cell receptor repertoires of lymphocytes found in kidney and blood samples of 10 patients with various renal diseases based on next-generation sequencing data. We used Biomed-2 primer panels and ImmunExplorer software to sequence, analyze and compare complementarity determining regions and V-(D)-J elements. While generally an individual’s renal receptor repertoire is different from the repertoire present in blood, 94% (30/32) of the lymphocytes with clonal expansion in kidney can also be traced in blood however, not all of these clonotypes are equally abundant. Summarizing the data of all analyzed patients, 68% of highly expanded T cell clonotypes and 30% of the highly expanded B cell clonotypes that have infiltrated the kidney can be found amongst the five most abundant clonotypes in blood. In addition, complementarity determining region 3 sequences of the immunoglobulin heavy chains are on average more diverse than T cell receptor beta chains. Immune repertoire analysis of tissue infiltrating B and T cells adds new approaches to the assessment of adaptive immune response in kidney diseases. Our data suggest that expanded clonotypes in the tissues might be traceable in blood samples in the course of treatment or the natural history of the disease.</p></div

CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.

<p>Percent values for expanded clonotypes in kidney and blood samples are highlighted in bold. Expanded clonotypes that could not be detected in the other compartment were marked as not detected, n.d. Clonotypes are also categorized according to primer sets regarding Biomed-2 primer panel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143125#pone.0143125.ref015" target="_blank">15</a>]. B cell results from patient 6 were discarded due to low amount of DNA input leading to inaccurate clonotype proportions. T cell results from blood sample of patient 10 were sorted in CD4⁺/CD8^- and CD4^-/CD8⁺ subpopulations. The T helper cell subpopulation showed no expansions and is therefore not listed (we found a very low abundance of the expanded clonotype in the kidney in this sample and we assume that these are traces from the CD4^-/CD8⁺ population and therefore this sample was discarded). Functionality of the rearranged chain is marked P for productive and UP for unproductive.</p><p>CDR3 sequences, V-(D)-J elements and functionality of highly expanded clonotypes in kidney and blood of all patients.</p

Quantification of element frequency (%) in highly expanded clonotypes.

<p>(A) VH, DH and JH element distribution of expanded B cell clonotypes and (B) Vß, Dß and Jß element distribution of expanded T cell clonotypes. We would appreciate the creation of a publicly available database with common V-(D)-J element frequency distribution in human populations to identify potential shifts in the course of diseases as to our knowledge this has not been provided to the community until this date.</p

Average percentage of blood and kidney lymphocyte subpopulations in all patients.

<p>T cells were separately analyzed for CD4⁺ and CD8⁺ subpopulations. NKT cells were gated using CD3⁺/CD56⁺ and NK cells, by CD3^-/CD56⁺. Cells presenting CD19⁺ and/or CD20⁺ surface markers were categorized as B cells. Significant differences were marked by a connecting line and the corresponding P-value (paired two-sided t-test).</p

Inverse Simpson’s diversity index and cell input.

<p>(a) IGH and TRB diversity of the average of all patients and healthy volunteers based on the inverse Simpson’s diversity index. Independent two-sided t test were performed with a level of significance of 0.05. (b) Average cell input of all patients and healthy volunteers for B and T cells. Paired two-sided t test were performed with a level of significance of 0.05. Error bars represent the standard deviation between samples of the same group.</p

Scientific Reports / Toxicological testing of allogeneic secretome derived from peripheral mononuclear cells (APOSEC): a novel cell-free therapeutic agent in skin disease

A cell-free approach using secretomes derived from stem cells or peripheral blood mononuclear cells is an active area of regenerative medicine that holds promise for therapies. Regulatory authorities classify these secretomes as biological medicinal products, and non- clinical safety assessment thus falls under the scope of ICH S6. A secretome of stressed peripheral blood mononuclear cells (APOSEC) was successfully tested in a toxicology program, supporting clinical use of the new drug candidate. Here, to allow for topical, dermal treatment of patients with diabetic foot ulcer, several non-clinical safety studies were performed. Acute toxicity (single dose) and neuropharmacological screening were tested intravenously in a rat model. Risk for skin sensitisation was tested in mice. A 4-week intravenous toxicity study in mice and a 4-week subcutaneous toxicity study in minipigs were conducted to cover the clinical setting and application in a rodent and a non-rodent model. Acute and repeated-dose toxicity studies show that APOSEC administered intravenously and subcutaneously does not involve major toxicities or signs of local intolerance at levels above the intended total human maximal dose of 3.3U/kg/treatment, 200U/wound/treatment, and 100U/cm/treatment. The non-clinical data support the safe topical use of APOSEC in skin diseases related to deficient wound healing.(VLID)493189

Safety and tolerability of topically administered autologous, apoptotic PBMC secretome (APOSEC) in dermal wounds : a randomized Phase 1 trial (MARSYAS I)

Developing effective therapies against chronic wound healing deficiencies is a global priority. Thus we evaluated the safety of two different doses of topically administered autologous APOSEC, the secretome of apoptotic peripheral blood mononuclear cells (PBMCs), in healthy male volunteers with artificial dermal wounds. Ten healthy men were enrolled in a single-center, randomized, double-blinded, placebo-controlled phase 1 trial. Two artificial wounds at the upper arm were generated using a 4-mm punch biopsy. Each participant was treated with both topically applied APOSEC and placebo in NuGel for 7 consecutive days. The volunteers were randomized into two groups: a low-dose group (A) receiving the supernatant of 12.5106 PBMCs and a high-dose group (B) receiving an equivalent of 25106 PBMCs resuspended in NuGel Hydrogel. Irradiated medium served as placebo. The primary outcome was the tolerability of the topical application of APOSEC. All adverse events were recorded until 17 days after the biopsy. Local tolerability assessment was measured on a 4-point scale. Secondary outcomes were wound closure and epithelization at day 7. No therapy-related serious adverse events occurred in any of the participants, and both low- and high-dose treatments were well tolerated. Wound closure was not affected by APOSEC therapy.(VLID)460739