Unraveling White Adipose Tissue Heterogeneity and Obesity by Adipose Stem/Stromal Cell Biology and 3D Culture Models.

The immune and endocrine dysfunctions of white adipose tissue are a hallmark of metabolic disorders such as obesity and type 2 diabetes. In humans, white adipose tissue comprises distinct depots broadly distributed under the skin (hypodermis) and as internal depots (visceral). Depot-specific ASCs could account for visceral and subcutaneous adipose tissue properties, by regulating adipogenesis and immunomodulation. More importantly, visceral and subcutaneous depots account for distinct contributions to obesity and its metabolic comorbidities. Recently, distinct ASCs subpopulations were also described in subcutaneous adipose tissue. Interestingly, the superficial layer closer to the dermis shows hyperplastic and angiogenic capacities, whereas the deep layer is considered as having inflammatory properties similar to visceral. The aim of this focus review is to bring the light of recent discoveries into white adipose tissue heterogeneity together with the biology of distinct ASCs subpopulations and to explore adipose tissue 3D models revealing their advantages, disadvantages, and contributions to elucidate the role of ASCs in obesity development. Recent advances in adipose tissue organoids opened an avenue of possibilities to recreate the main cellular and molecular events of obesity leading to a deep understanding of this inflammatory disease besides contributing to drug discovery. Furthermore, 3D organ-on-a-chip will add reproducibility to these adipose tissue models contributing to their translation to the pharmaceutical industry.

Bone marrow-derived mesenchymal stem cells transplantation ameliorates renal injury through anti-fibrotic and anti-inflammatory effects in chronic experimental renovascular disease.

BACKGROUND: Progressive renal fibrosis is an underlying pathological process of chronic kidney disease (CKD) evolution. This study aimed to evaluate the roles of bone-marrow-derived mesenchymal stem cells (MSC) in the remodeling of fibrotic kidney parenchyma in the two kidneys-one clip (2K1C) CKD animal model. METHODS: Wistar rats were allocated into three groups: Sham, 2K1C, and 2K1C þ MSC. MSCs (106) were transplanted into the renal subcapsular region two weeks after clipping the left renal artery. Six weeks after clipping, left kidney samples were analyzed using histological and western blotting techniques. ANOVA tests were performed and differences between groups were considered statistically significant if p < 0.05. RESULTS: Clipped kidneys of 2K1C rats displayed renal fibrosis, with excessive collagen deposition, glomerulosclerosis and renal basement membrane disruption. Clipped kidneys of 2K1C þ MSC rats showed preserved Bowman's capsule and tubular basement membranes, medullary tubules morphological reconstitution and reduced collagen deposits. Expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 were elevated, whereas tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 levels were decreased in clipped kidneys of 2K1C rats. MSCs transplantation restored these expression levels. Moreover, MSCs suppressed macrophages and myofibroblasts accumulation, as well as TNF-a expression in clipped kidneys of 2K1C animals. MSCs transplantation significantly increased IL-10 expression. CONCLUSIONS: Transplanted MSCs orchestrate anti-fibrotic and anti-inflammatory events, which reverse renal fibrosis and promote renal morphological restoration. This study supports the notion that only one MSCs delivery into the renal subcapsular region represents a possible therapeutic strategy against renal fibrosis for CKD treatment.

Altered Umbilical Cord Blood Nutrient Levels, Placental Cell Turnover and Transporter Expression in Human Term Pregnancies Conceived by Intracytoplasmic Sperm Injection (ICSI).

Assisted reproductive technologies (ART) may increase risk for abnormal placental development, preterm delivery and low birthweight. We investigated placental morphology, transporter expression and paired maternal/umbilical fasting blood nutrient levels in human term pregnancies conceived naturally (n = 10) or by intracytoplasmic sperm injection (ICSI; n = 11). Maternal and umbilical vein blood from singleton term (>37 weeks) C-section pregnancies were assessed for levels of free amino acids, glucose, free fatty acids (FFA), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low-density lipoprotein (VLDL) and triglycerides. We quantified placental expression of GLUT1 (glucose), SNAT2 (amino acids), P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) (drug) transporters, and placental morphology and pathology. Following ICSI, placental SNAT2 protein expression was downregulated and umbilical cord blood levels of citrulline were increased, while FFA levels were decreased at term (p < 0.05). Placental proliferation and apoptotic rates were increased in ICSI placentae (p < 0.05). No changes in maternal blood nutrient levels, placental GLUT1, P-gp and BCRP expression, or placental histopathology were observed. In term pregnancies, ICSI impairs placental SNAT2 transporter expression and cell turnover, and alters umbilical vein levels of specific nutrients without changing placental morphology. These may represent mechanisms through which ICSI impacts pregnancy outcomes and programs disease risk trajectories in offspring across the life course.

Therapeutic potential of mesenchymal stem cells in multiple organs affected by COVID-19.

Currently, the world has been devastated by an unprecedented pandemic in this century. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the agent of coronavirus disease 2019 (COVID-19), has been causing disorders, dysfunction and morphophysiological alterations in multiple organs as the disease evolves. There is a great scientific community effort to obtain a therapy capable of reaching the multiple affected organs in order to contribute for tissue repair and regeneration. In this regard, mesenchymal stem cells (MSCs) have emerged as potential candidates concerning the promotion of beneficial actions at different stages of COVID-19. MSCs are promising due to the observed therapeutic effects in respiratory preclinical models, as well as in cardiac, vascular, renal and nervous system models. Their immunomodulatory properties and secretion of paracrine mediators, such as cytokines, chemokines, growth factors and extracellular vesicles allow for long range tissue modulation and, particularly, blood-brain barrier crossing. This review focuses on SARS-CoV-2 impact to lungs, kidneys, heart, vasculature and central nervous system while discussing promising MSC's therapeutic mechanisms in each tissue. In addition, MSC's therapeutic effects in high-risk groups for COVID-19, such as obese, diabetic and hypertensive patients are also explored.

Predisposition to acute pancreatitis in dogs with severe acute renal failure / Predisposição à pancreatite aguda em cães com insuficiência renal aguda grave

Patients with acute renal failure often have a negative energy balance, which promotes metabolic changes predisposing to complications. The objective of this study was to evaluate laboratory parameters of 30 dogs with severe acute renal failure, to assess their relationship with the possibility of developing acute pancreatitis due to the negative energy balance, and to correlate these findings with the degree of renal failure. Serum concentrations of urea, creatinine, beta-hydroxybutyrate, triglycerides, amylase, total lipase, and canine pancreatic lipase were compared between healthy dogs and dogs with severe acute renal failure. A greater serum concentration of beta-hydroxybutyrate and greater activity of pancreatic enzymes, particularly canine pancreatic lipase, as well as a stronger correlation between the latter and serum creatinine concentrations, were related to the occurrence of acute pancreatitis in patients with severe acute renal failure. A greater degree of renal failure implied a greater predisposition to acute pancreatitis.(AU)

O portador de insuficiência renal aguda é um paciente que, muitas vezes, encontra-se sob importante condição de balanço energético negativo, gerando alterações metabólicas que predispõem a complicações. O objetivo deste estudo foi avaliar parâmetros laboratoriais de trinta cães com insuficiência renal aguda grave, quanto a possibilidade de desenvolvimento de pancreatite aguda em função do balanço energético negativo, e relacioná-los ao grau de gravidade da insuficiência renal. As concentrações séricas de ureia, creatinina, betahidroxibutirato, triglicérides, amilase, lipase total e lipase pancreática canina foram comparadas entre o grupo de cães hígidos e o de cães doentes. Observou-se maior concentração sérica de betahidroxibutirato e maior atividade das enzimas pancreáticas, especialmente da lipase pancreática canina, além de forte correlação entre esta última e a concentração sérica de creatinina, demonstrando a ocorrência de pancreatite aguda em pacientes com insuficiência renal aguda grave. Verificou-se também que quanto mais grave é a insuficiência renal, maior é a predisposição à pancreatite aguda.(AU)

Predisposition to acute pancreatitis in dogs with severe acute renal failure

ABSTRACT: Patients with acute renal failure often have a negative energy balance, which promotes metabolic changes predisposing to complications. The objective of this study was to evaluate laboratory parameters of 30 dogs with severe acute renal failure, to assess their relationship with the possibility of developing acute pancreatitis due to the negative energy balance, and to correlate these findings with the degree of renal failure. Serum concentrations of urea, creatinine, beta-hydroxybutyrate, triglycerides, amylase, total lipase, and canine pancreatic lipase were compared between healthy dogs and dogs with severe acute renal failure. A greater serum concentration of beta-hydroxybutyrate and greater activity of pancreatic enzymes, particularly canine pancreatic lipase, as well as a stronger correlation between the latter and serum creatinine concentrations, were related to the occurrence of acute pancreatitis in patients with severe acute renal failure. A greater degree of renal failure implied a greater predisposition to acute pancreatitis.

RESUMO: O portador de insuficiência renal aguda é um paciente que, muitas vezes, encontra-se sob importante condição de balanço energético negativo, gerando alterações metabólicas que predispõem a complicações. O objetivo deste estudo foi avaliar parâmetros laboratoriais de trinta cães com insuficiência renal aguda grave, quanto a possibilidade de desenvolvimento de pancreatite aguda em função do balanço energético negativo, e relacioná-los ao grau de gravidade da insuficiência renal. As concentrações séricas de ureia, creatinina, betahidroxibutirato, triglicérides, amilase, lipase total e lipase pancreática canina foram comparadas entre o grupo de cães hígidos e o de cães doentes. Observou-se maior concentração sérica de betahidroxibutirato e maior atividade das enzimas pancreáticas, especialmente da lipase pancreática canina, além de forte correlação entre esta última e a concentração sérica de creatinina, demonstrando a ocorrência de pancreatite aguda em pacientes com insuficiência renal aguda grave. Verificou-se também que quanto mais grave é a insuficiência renal, maior é a predisposição à pancreatite aguda.

Allogenic Adipose Tissue-Derived Stromal/Stem Cells and Vitamin D Supplementation in Patients With Recent-Onset Type 1 Diabetes Mellitus: A 3-Month Follow-Up Pilot Study.

Objective: To evaluate the short term safety and potential therapeutic effect of allogenic adipose tissue-derived stromal/stem cells (ASCs) + cholecalciferol in patients with recent-onset T1D. Methods: Prospective, phase II, open trial, pilot study in which patients with recent onset T1D received ASCs (1 × 106 cells/kg) and cholecalciferol 2000 UI/day for 3 months (group 1) and were compared to controls with standard insulin therapy (group 2). Adverse events, C-peptide (CP), insulin dose, HbA1c, time in range (TIR), glucose variability (continuous glucose monitoring) and frequency of CD4+FoxP3+ T-cells (flow cytometry) were evaluated at baseline (T0) and after 3 months (T3). Results: 13 patients were included (8: group 1; 5: group 2). Their mean age and disease duration were 26.7 ± 6.1 years and 2.9 ± 1.05 months. Adverse events were transient headache (n = 8), mild local reactions (n = 7), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), mild floaters (n = 2), central retinal vein occlusion (n = 1, complete resolution). At T3, group 1 had lower insulin requirement (0.22 ± 0.17 vs. 0.61±0.26IU/Kg; p = 0.01) and HbA1c (6.47 ± 0.86 vs. 7.48 ± 0.52%; p = 0.03) than group 2. In group 1, 2 patients became insulin free (for 4 and 8 weeks) and all were in honeymoon at T3 (vs. none in group 2; p = 0.01). CP variations did not differ between groups (-4.6 ± 29.1% vs. +2.3 ± 59.65%; p = 0.83). Conclusions: Allogenic ASCs + cholecalciferol without immunosuppression was associated with stability of CP and unanticipated mild transient adverse events in patients with recent onset T1D. ClinicalTrials.gov registration: NCT03920397.

Biologically produced silver chloride nanoparticles from B. megaterium modulate interleukin secretion by human adipose stem cell spheroids.

Stem cell tissue constructs are likely to come into contact with silver-based nanoparticles-such as silver chloride nanoparticles (AgCl-NPs)-used as microbicidals at the implant site or in cosmetics. However, the effect of silver-based nanoparticles on 3D cell cultures with potential for tissue engineering has received little attention. Here, we examined the effect of sub-lethal doses (5, 10 and 25 µg/mL, for 1, 7 and 21 days) of AgCl-NPs produced by 'green' bacterial-based synthesis on spheroid 3D cultures of human adipose tissue stem cells (ASCs). Light microscopy analysis revealed that the shape and diameter of ASC spheroids remained largely unchanged after AgCl-NP treatment. Flow cytometry analysis with 7-AAD and 2',7'-dichlorofluorescein diacetate revealed no statistically significant differences in cell death but showed an increase of ROS levels for the untreated group and significant differences for the groups treated with 5 and 10 µg/mL at day 7 (p = 0.0395, p = 0.0266, respectively). Electron microscopy analysis showed limited cell damage in the periphery of AgCl-NP-treated spheroids. However, treatment with AgCl-NP had statistically significant effects on the secretion of IL-6, IL-8, IL-1ß and IL-10 by spheroids, at specific treatment periods and concentrations, and particularly for IL-6, IL-8 and IL-1ß. TGF-ß1 and -ß2 secretion also changed significantly throughout the treatment period. Our results indicate that, despite having little effect on cell viability and morphology, sub-lethal AgCL-NP doses modulate ROS production at day 7 for the groups treated with 5 and 10 µg/mL and also modulate the secretory profile of ASC spheroids. Thus, the use of skin implants or products containing Ag-NPs may promote long-term disturbances in subcutaneous adipose tissue homeostasis.

Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel.

The scaffold-free tissue engineering using spheroids is pointed out as an approach for optimizing the delivery system of cartilage construct. In this study, we aimed to evaluate the micromolded nonadhesive hydrogel (MicroTissues®) for spheroid compaction (2-day culture) and spontaneous chondrogenesis (21-day culture) using cartilage progenitors cells (CPCs) from human nasal septum without chondrogenic stimulus. CPC spheroids showed diameter stability (486 µm ± 65), high percentage of viable cells (88.1 ± 2.1), and low percentage of apoptotic cells (2.3%). After spheroid compaction, the synthesis of TGF-ß1, TGF-ß2, and TGF-ß3 was significantly higher compared to monolayer (p < 0.005). Biomechanical assay revealed that the maximum forces applied to spheroids after chondrogenesis were 2.6 times higher than for those cultured for 2 days. After spontaneous chondrogenesis, CPC spheroids were entirely positive for N-cadherin, collagen type II and type VI, and aggrecan and chondroitin sulfate. Comparing to monolayer, the expression of SOX5 and SOX6 genes analyzed by qPCR was significantly upregulated (p < 0.01). Finally, we observed the capacity of CPC spheroids starting to fuse. To the best of our knowledge, this is the first time in the scientific literature that human CPC spheroids were formed by micromolded nonadhesive hydrogel, achieving a successful scaffold-free cartilage engineering without chondrogenic stimulus (low cost).

Delivery of Human Adipose Stem Cells Spheroids into Lockyballs.

Adipose stem cells (ASCs) spheroids show enhanced regenerative effects compared to single cells. Also, spheroids have been recently introduced as building blocks in directed self-assembly strategy. Recent efforts aim to improve long-term cell retention and integration by the use of microencapsulation delivery systems that can rapidly integrate in the implantation site. Interlockable solid synthetic microscaffolds, so called lockyballs, were recently designed with hooks and loops to enhance cell retention and integration at the implantation site as well as to support spheroids aggregation after transplantation. Here we present an efficient methodology for human ASCs spheroids biofabrication and lockyballs cellularization using micro-molded non-adhesive agarose hydrogel. Lockyballs were produced using two-photon polymerization with an estimated mechanical strength. The Young's modulus was calculated at level 0.1362 +/-0.009 MPa. Interlocking in vitro test demonstrates high level of loading induced interlockability of fabricated lockyballs. Diameter measurements and elongation coefficient calculation revealed that human ASCs spheroids biofabricated in resections of micro-molded non-adhesive hydrogel had a more regular size distribution and shape than spheroids biofabricated in hanging drops. Cellularization of lockyballs using human ASCs spheroids did not alter the level of cells viability (p > 0,999) and gene fold expression for SOX-9 and RUNX2 (p > 0,195). The biofabrication of ASCs spheroids into lockyballs represents an innovative strategy in regenerative medicine, which combines solid scaffold-based and directed self-assembly approaches, fostering opportunities for rapid in situ biofabrication of 3D building-blocks.

Stromal-vascular fraction content and adipose stem cell behavior are altered in morbid obese and post bariatric surgery ex-obese women.

INTRODUCTION: Subcutaneous adipose tissue is an interesting source of autologous stem cells with a fundamental role in the pathophysiology of obesity, metabolic syndromes and insulin resistance. We hypothesize that obesity could alter the stromal-vascular fraction (SVF) and adipose stem cell (ASCs) functions, which could compromise its regenerative behavior. Furthermore, we aimed to evaluate whether ASCs derived from post bariatric surgery ex-obese women maintain their functions in a similar fashion as do those from individuals who have never been obese. METHODS: The SVF of subcutaneous adipose tissue from control (n = 6, body mass index - BMI - 27.5 ± 0.5 kg/m(2)), obese (n = 12, BMI 46.2 ± 5.1 kg/m(2)) and post bariatric surgery ex-obese (n = 7, initial BMI 47.8 ± 1.3 kg/m(2); final BMI 28.1 ± 1.1 kg/m(2)) women were isolated and evaluated by flow cytometry. ASCs were tested for lipid accumulation by perilipin, adipose differentiation-related protein (ADRP) and Oil Red O staining after adipogenic stimulus. The cytokines secreted by the ASCs and after lipid accumulation induction were also evaluated. RESULTS: The subcutaneous adipose tissue of obese and post bariatric surgery ex-obese women was enriched in pericytes (p = 0.0345). The number of supra-adventitial cells was not altered in the obese patients, but it was highly enriched in the post bariatric surgery ex-obese women (p = 0.0099). The ASCs of the post bariatric surgery ex-obese patients secreted more MCP-1 (monocyte chemoattractant protein-1; p = 0.0078). After lipid accumulation induction, the ASCs of the patients in all groups secreted less IL-6 than the ASCs with no adipogenic stimulus (p < 0.0001). Obese ASCs with lipid accumulation secreted the highest amount of IL-6 (p < 0.001) whereas the ASCs from the controls secreted the highest amount of adiponectin (p < 0.0001). The ASCs from the post bariatric surgery ex-obese patients showed the highest levels of lipid accumulation whereas those from the obese women had the lowest levels (p < 0.0001). CONCLUSIONS: SVF content and ASC behavior are altered in the subcutaneous adipose tissue of morbid obese women; these changes are not completely restored after bariatric surgery-induced weight loss. The cellular alterations described in this study could affect the regenerative effects of adipose stem cells. Further investigations are required to avoid jeopardizing the development of autologous stem cell-based therapies.

Obesity and weight loss could alter the properties of adipose stem cells?

The discovery that adipose tissue represents an interesting source of multipotent stem cells has led to many studies exploring the clinical potential of these cells in cell-based therapies. Recent advances in understanding the secretory capacity of adipose tissue and the role of adipokines in the development of obesity and associated disorders have added a new dimension to the study of adipose tissue biology in normal and diseased states. Subcutaneous adipose tissue forms the interface between the clinical application of regenerative medicine and the establishment of the pathological condition of obesity. These two facets of adipose tissue should be understood as potentially related phenomena. Because of the functional characteristics of adipose stem cells, these cells represent a fundamental tool for understanding how these two facets are interconnected and could be important for therapeutic applications. In fact, adipose tissue stem cells have multiple functions in obesity related to adipogenic, angiogenic and secretory capacities. In addition, we have also previously described a predominance of larger blood vessels and an adipogenic memory in the subcutaneous adipose tissue after massive weight loss subsequent to bariatric surgery (ex-obese patients). Understanding the reversibility of the behavior of adipose stem cells in obeses and in weight loss is relevant to both physiological studies and the potential use of these cells in regenerative medicine.

Bioengineered cartilage in a scaffold-free method by human cartilage-derived progenitor cells: a comparison with human adipose-derived mesenchymal stromal cells.

The objective of our study was to investigate chondrogenesis potential of human adipose-derived mesenchymal stromal cells (MSCs), using as a positive control a human source of cartilage-derived progenitor cells (PCs). This source of PCs was recently described by our group and dwells on the surface of nasoseptal cartilage. Histological analysis using Safranin O staining and immunofluorescence for actin filaments and collagen type II was performed on three-dimensional (3D) pellet cultures. Cartilage PCs and adipose MSCs showed similarities in monolayer culture related to cell morphology and proliferation. Our 3D pellet cultures substantially reduced the actin stress and after 21 days under chondrogenic medium, we observed an increase in the pellet diameter for cartilage PCs (7.4%) and adipose MSCs (21.2%). Adipose-derived MSCs responded to chondrogenic stimulus, as seen by positive areas for collagen type II, but they were not able to recreate a mature extracellular matrix. Using semi-quantitative analysis, we observed a majority of Safranin O areas rising from blue (no stain) to orange (moderate staining) and no changes in fibroblastic morphology (P < 0.0001). For cartilage PCs, chondrogenic induction is responsible for morphological changes and a high percentage of matrix area/number of cells (P ≤ 0.0001), evaluated by computerized histomorphometry. Morphological analyses reveal that adipose-derived MSCs were not able to recreate a bioengineered cartilage. The cost of culture was reduced, as the cartilage PCs under growth-factor free medium exhibit a high score for cartilage formation compared with the induced adipose mesenchymal stromal cells (P = 0.0021). Using a pellet 3D culture, our cartilage PCs were able to produce a cartilage tissue in vitro, leading to the future development of bioengineered products.

Isolation of human nasoseptal chondrogenic cells: a promise for cartilage engineering.

In cartilaginous tissues, perichondrium cambium layer may be the source of new cartilage. Human nasal septal perichondrium is considered to be a homogeneous structure in which some authors do not recognize the perichondrium internal zone or the cambium layer as a layer distinct from adjacent cartilage surface. In the present study, we isolated a chondrogenic cell population from human nasal septal cartilage surface zone. Nasoseptal chondrogenic cells were positive for surface markers described for mesenchymal stem cells, with exception of CD146, a perivascular cell marker, which is consistent with their avascular niche in cartilage. Although only Sox-9 was constitutively expressed, they also revealed osteogenic and chondrogenic, but not adipogenic, potentials in vitro, suggesting a more restricted lineage potential compared to mesenchymal stem cells. Interestingly, even in absence of chondrogenic growth factors in the pellet culture system, nasoseptal chondrogenic cells had a capacity to synthesize sulfated glycosaminoglycans, large amounts of collagen type II and to a lesser extent collagen type I. The spontaneous chondrogenic potential of this population of cells indicates that they may be a possible source for cartilage tissue engineering. Besides, the pellet culture system using nasoseptal chondrogenic cells may also be a model for studies of chondrogenesis.