Evaluation of type 1 diabetes' partial clinical remission after three years of heterologous adipose tissue derived stromal/stem cells transplantation associated with vitamin D supplementation.

BACKGROUND: Mesenchymal stem cell infusion and vitamin D supplementation may have immunomodulatory actions that could prolong the preservation of residual insulin secretion in patients with type 1 diabetes (T1D). Intervention with these agents after onset of T1D could favor the development of a remission phase, with potential clinical impact. We aimed to compare the presence of clinical remission (CR), glycemic control and daily insulin requirement at 6, 12, 18, 24 and 36 months after the diagnosis of T1D using IDAA1c in patients who received therapy with adipose tissue-derived mesenchymal stem cell (ASC) infusion and vitamin D supplementation and a control group. METHODS: This retrospective cohort study analyzed data from the medical records of patients with T1D diagnosed between 15 and 40 years. Partial CR was defined as an IDAA1c index < 9. Patients in the intervention group received an infusion of adipose tissued-derived mesenchymal stem cells (ASCs) within 3 months after diagnosis and supplementation with 2000 IU of cholecalciferol for 1 year, started on the day following the infusion. Partial CR was also determined using the ISPAD criteria, to assess its agreement with IDAA1c. RESULTS: A total of 28 patients were evaluated: 7 in the intervention group (group 1) and 21 in the control group (group 2). All patients in group 1 evolved with partial CR while only 46.7% of patients in group 2 had this outcome. Group 1 had a higher frequency of CR when evaluated with IDAA1c and ISPAD criteria. The mean duration of CR varied between the two criteria. Although HbA1c was similar between groups during follow-up, group 1 had a lower total daily insulin requirement (p < 0.005) at all time points. At 36 months, group 1 used 49% of the total daily insulin dose used by group 2 with similar glycemic control. CONCLUSION: The intervention with infusion of ASC + vitamin D supplementation was associated with partial CR at 6 months. Although there were no differences in CR established by the IDAA1c and ISPAD criteria after three years of follow-up, patients who underwent intervention had nearly the half insulin requirement of controls with conventional treatment, with similar glycemic control. TRIAL REGISTRATION: 37001514.0.0000.5257.

High Concentrations of Cannabidiol Induce Neurotoxicity in Neurosphere Culture System.

Recent studies have demonstrated that cannabinoids are potentially effective in the treatment of various neurological conditions, and cannabidiol (CBD), one of the most studied compounds, has been proposed as a non-toxic option. However, the adverse effects of CBD on neurodevelopmental processes have rarely been studied in cell culture systems. To better understand CBD's influence on neurodevelopment, we exposed neural progenitor cells (NPCs) to different concentrations of CBD (1 µM, 5 µM, and 10 µM). We assessed the morphology, migration, differentiation, cell death, and gene expression in 2D and 3D bioprinted models to stimulate physiological conditions more effectively. Our results showed that CBD was more toxic at higher concentrations (5 µM and 10 µM) and affected the viability of NPCs than at lower concentrations (1 µM), in both 2D and 3D models. Moreover, our study revealed that higher concentrations of CBD drastically reduced the size of neurospheres and the number of NPCs within neurospheres, impaired the morphology and mobility of neurons and astrocytes after differentiation, and reduced neurite sprouting. Interestingly, we also found that CBD alters cellular metabolism by influencing the expression of glycolytic and ß-oxidative enzymes in the early and late stages of metabolic pathways. Therefore, our study demonstrated that higher concentrations of CBD promote important changes in cellular functions that are crucial during CNS development.

Potential and Limitations of Induced Pluripotent Stem Cells-Derived Mesenchymal Stem Cells in Musculoskeletal Disorders Treatment.

The burden of musculoskeletal disorders (MSK) is increasing worldwide. It affects millions of people worldwide, decreases their quality of life, and can cause mortality. The treatment of such conditions is challenging and often requires surgery. Thus, it is necessary to discuss new strategies. The therapeutic potential of mesenchymal stem cells (MSC) in several diseases has been investigated with relative success. However, this potential is hindered by their limited stemness and expansion ability in vitro and their high donor variability. MSC derived from induced pluripotent stem cells (iPSC) have emerged as an alternative treatment for MSK diseases. These cells present distinct features, such as a juvenile phenotype, in addition to higher stemness, proliferation, and differentiation potential than those of MSC. Here, we review the opportunities, challenges, and applications of iPSC as relevant clinical therapeutic cell sources for MSK disorders. We discuss iPSC sources from which to derive iMSC and the advantages and disadvantages of iMSC over MSC as a therapeutic approach. We further summarize the main preclinical and clinical studies exploring the therapeutic potential of iMSC in MSK disorders.

Adipose Tissue-Derived Stromal/Stem Cells Transplantation with Cholecalciferol Supplementation in Recent-Onset Type 1 Diabetes Patients: Twelve Months Follow-Up.

To evaluate safety and therapeutic effect along 12 months of allogenic adipose tissue-derived stromal/stem cells (ASCs) transplantation with cholecalciferol (VITD) in patients with recent-onset type 1 diabetes (T1D). Prospective, phase II, open trial, pilot study in which patients with recent onset T1D received ASCs (1xKgx106 cells) and VITD 2000UI/day for 12 months (group 1) and were compared to controls with standard insulin therapy (group 2). Adverse events, C-peptide area under the curve (CPAUC), insulin dose, HbA1c and frequency of FoxP3+ in CD4+ or CD8+ T-cells(flow cytometry) were evaluated at baseline(T0), after 3(T3), 6(T6) and 12 months(T12). Eleven patients completed follow up (7:group 1;4:group 2). Group 1 had lower insulin requirement at T3(0.24±0.18vs0.53±0.23UI/kg,p=0.04), T6(0.24±0.15vs0.66±0.33 UI/kg,p=0.04) and T12(0.39±0.15vs0.74±0.29 UI/Kg,p=0.04).HbA1c was lower at T6 (50.57±8.56vs72.25±10.34 mmol/mol,p=0.01), without differences at T12 (57.14±11.98 in group 1 vs. 73.5±14.57 mmol/min in group 2, p=0.16). CPAUC was not significantly different between groups at T0(p=0.07), higher in group 1 at T3(p=0.04) and T6(p=0.006), but similar at T12(p=0.23). IDAA1c was significantly lower in group 1 than group 2 at T3,T6 and T12 (p=0.006, 0.006 and 0.042, respectively). IDDA1c was inversely correlated to FoxP3 expression in CD4 and CD8+ T cells at T6 (p<0.001 and p=0.01, respectively). In group 1, one patient had recurrence of a benign teratoma that was surgically removed, not associated to the intervention. ASCs with VITD without immunosuppression were safe and associated lower insulin requirements, better glycemic control, and transient better pancreatic function in recent onset T1D, but the potential benefits were not sustained.

Seroprevalence to Rabies Virus in Wildlife in Brazil.

Serum samples of 638 free-ranging wild mammals from São Paulo state, Brazil, were tested for neutralizing antibodies against rabies virus by the rapid fluorescent focus inhibition test. Overall seroprevalence was 1.7% among 24 species surveyed, with individuals of six species having positive results indicating exposure to rabies virus.

Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients.

OBJECTIVE: Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. METHODS: In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 × 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). RESULTS: In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. CONCLUSION: Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of ß-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P < .0001), IL-8 (P < .0001), IL-10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

Descrição do comportamento reprodutivo de Bothrops pauloensis (Serpentes, Viperidae) em cativeiro / Description of the reproductive behavior of Bothrops pauloensis (Serpentes, Viperidae) in captivity

Snakes is carried out through random field records, studies of specimens from scientific collections and data generated in captivity. The maintenance of venomous snakes in captivity at the Butantan Institute is important due to the production of immunobiologicals and toxinology research at the Institute or at other research centers. The black faced lancehead (Bothrops pauloensis) is an endemic viper of the Brazilian cerrado, present in seasonally dry open savannas. This study aimed to describe the behavior of courtship and copulation of B. pauloensis in captivity. During the months of June to August in 2019, seven copulation attempts were made, in which the behaviors of couples of trios of animals (one female and two males) were recorded in a breeding arena (115x65x60 cm). The female was placed in the arena 30 minutes after the presence of the males. The next day, the animals were separated, from the arena and then the female to perform the vaginal secretion collection (exfoliative cytology) (an examination where material is collected from the female's vagina, smearing and staining the slides and as well as the elaboration of photomicrographs to check for the presence of sperm. Three females obtained positive swabs with sperm on the slides. During the court, one of the males became interested in the female to do the reconnaissance and stimulate the copulation, the other male eventually moved away and hid under the cardboard in the arena. During the court, "head-rise", "mount" and "tail quiver" movements were observed. These mating behaviors are typical and have been described in other neotropical viperids, such as B. erythromelas. In short, B. pauloensis exhibited ritualistic behavior at the time of reproduction similar to that described for other Bothrops. The best period for copulation was during the month of July, during which the females were more receptive.

O estudo da biologia reprodutiva de serpentes é realizado através de registros fortuitos em campo, estudos de espécimes de coleções científicas e de dados gerados em cativeiro. A manutenção de serpentes peçonhentas em cativeiro do Instituto Butantan é importante devido à produção de imunobiológicos e de pesquisas em toxinologia no Instituto ou em outros centros de pesquisa. A jararaca- pintada (Bothrops pauloensis) é um viperídeo endêmico do cerrado brasileiro, presente em savanas abertas sazonalmente secas. Este estudo teve como objetivo descrever o comportamento de corte e cópula de Bothrops pauloensis em cativeiro. Durante os meses de junho a agosto de 2019, foram realizadas sete tentativas de cópula, nas quais os comportamentos dos casais de trios de animais (uma fêmea e dois machos) foram registrados em uma arena de reprodução (115x65x60 cm). A fêmea foi colocada na arena 30 minutos após a presença dos machos. No dia seguinte, os machos eram retirados da arena e depois a fêmea para a realização da coleta da secreção vaginal (citologia esfoliativa) (um exame onde se colhe material da vagina da fêmea, fazendo um esfregaço e coloração das lâminas e assim como elaboração das fotomicrografias para verificação da presença de espermatozoides. Três fêmeas obtiveram exames positivos apresentando espermatozoides. Durante a corte um dos machos se interessou pela fêmea para fazer o reconhecimento e estimular para a cópula, o outro macho eventualmente afastou-se e escondeu-se de baixo do papelão na arena. Durante a corte, foram observados movimentos de "subida da cabeça", "montagem" e "tremor de cauda". Esses comportamentos de acasalamento são típicos e já foram descritos em outros viperídeos neotropicais, como B. erythromelas. Em suma, B. pauloensis exibiu comportamento ritualístico na época de reprodução semelhante ao descrito para outras Bothrops. O melhor período para cópula foi durante o mês de julho, período que as fêmeas se apresentaram mais receptivas.

Neuromedin B receptor disruption impairs adipogenesis in mice and 3T3-L1 cells.

Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33-39%), reduced cell proliferation at day 3 (-53%) and reduced lipid accumulation at day 21 (-86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.

Adipose-derived stromal/stem cells from different adipose depots in obesity development.

The increasing prevalence of obesity is alarming because it is a risk factor for cardiovascular and metabolic diseases (such as type 2 diabetes). The occurrence of these comorbidities in obese patients can arise from white adipose tissue (WAT) dysfunctions, which affect metabolism, insulin sensitivity and promote local and systemic inflammation. In mammals, WAT depots at different anatomical locations (subcutaneous, preperitoneal and visceral) are highly heterogeneous in their morpho-phenotypic profiles and contribute differently to homeostasis and obesity development, depending on their ability to trigger and modulate WAT inflammation. This heterogeneity is likely due to the differential behavior of cells from each depot. Numerous studies suggest that adipose-derived stem/stromal cells (ASC; referred to as adipose progenitor cells, in vivo) with depot-specific gene expression profiles and adipogenic and immunomodulatory potentials are keys for the establishment of the morpho-functional heterogeneity between WAT depots, as well as for the development of depot-specific responses to metabolic challenges. In this review, we discuss depot-specific ASC properties and how they can contribute to the pathophysiology of obesity and metabolic disorders, to provide guidance for researchers and clinicians in the development of ASC-based therapeutic approaches.

Adaptation of a skin sensitization assay to a chemically defined culture.

There are currently three in vitro methods adopted by the Organization for the Economic Co-operation and Development for testing chemicals based on the third key event of the skin sensitization adverse outcome pathway, the activation of dendritic cells. All of them use culture medium supplemented with fetal bovine serum (FBS), which brings technical disadvantages and animal welfare concerns. The objective of this study was to analyze the possibility of eliminating the use of FBS in the human Cell Line Activation Test (h-CLAT). After successful implementation of the h-CLAT using THP-1 cells cultured in FBS-containing medium, several attempts to adapt THP-1 cells to four different serum-free media were made. The best results were obtained with gradual adaptation to RPMI-1640 medium with HL-1™ Supplement and to X-VIVO™ 10. Adapted cells were cryopreserved and submitted to the reactivity check. After being approved, they were used in dose finding and proficiency assays. Despite minor adjustments in the original protocol, it was possible to correctly predict the sensitizing potential of the ten proficiency substances using THP-1 cells adapted to X-VIVO™ 10, which indicates that it is possible to eliminate the use of FBS in the h-CLAT, using a chemically defined medium.

Predictive factors of weaning from mechanical ventilation and extubation outcome: A systematic review.

PURPOSE: To identify, describe and discuss the parameters used to predict weaning from mechanical ventilation and extubation outcomes. METHODS: Systematic review of scientific articles using four electronic databases: PubMed, Embase, PEDro and Cochrane Library. Search terms included "weaning", "extubation", "withdrawal" and "discontinuation", combined with "mechanical ventilation" and "predictive factors", "predictive parameters" and "predictors for success". In this study, we included original articles that presented predictive factors for weaning or extubation outcomes in adult patients and not restricted to a single disease. Articles not written in English were excluded. RESULTS: A total of 43 articles were included, with a total of 7929 patients and 56 different parameters related to weaning and extubation outcomes. Rapid Shallow Breathing Index (RSBI) was the most common predictor, discussed in 15 studies (2159 patients), followed by Age and Maximum Inspiratory Pressure in seven studies. The other 53 parameters were found in less than six studies. CONCLUSION: There are several parameters used to predict weaning and extubation outcomes. RSBI was the most frequently studied and seems to be an important measurement tool in deciding whether to wean/extubate a patient. Furthermore, the results demonstrated that weaning and extubation should be guided by several parameters, and not only to respiratory ones.

Spheroids of stem cells as endochondral templates for improved bone engineering.

Osteodegenerative disease and bone fractures lead to bone damage or loss, requiring new bone formation to replace the damaged tissues. Classical 'top-down' tissue engineering relies on seeding cell suspensions into biomaterial scaffolds, and then guiding cell fate by growth factors. However, complex tissue fabrication using this approach has important limitations. 'Bottom-up' tissue engineering has the potential to overcome the drawbacks of the top-down approach, by using 'building blocks' of cell spheroids for tissue biofabrication without a scaffold. Spheroids are 3D structures that resemble the physiological tissue microenvironment and can be produced in vitro by different methods. Spheroids of mesenchymal stem cells (MSC) and adipose stem cells (ASC) have regenerative properties. Here we review, the use of spheroids as 'building blocks' in the 3D bioprinting of large-scale bone tissue and as a promising alternative for the treatment of osteodegenerative diseases and in bone engineering, including endochondral ossification (or developmental engineering).

Characterization of stromal vascular fraction and adipose stem cells from subcutaneous, preperitoneal and visceral morbidly obese human adipose tissue depots.

BACKGROUND/OBJECTIVES: The pathological condition of obesity is accompanied by a dysfunctional adipose tissue. We postulate that subcutaneous, preperitoneal and visceral obese abdominal white adipose tissue depots could have stromal vascular fractions (SVF) with distinct composition and adipose stem cells (ASC) that would differentially account for the pathogenesis of obesity. METHODS: In order to evaluate the distribution of SVF subpopulations, samples of subcutaneous, preperitoneal and visceral adipose tissues from morbidly obese women (n = 12, BMI: 46.2±5.1 kg/m2) were collected during bariatric surgery, enzymatically digested and analyzed by flow cytometry (n = 12). ASC from all depots were evaluated for morphology, surface expression, ability to accumulate lipid after induction and cytokine secretion (n = 3). RESULTS: A high content of preadipocytes was found in the SVF of subcutaneous depot (p = 0.0178). ASC from the three depots had similar fibroblastoid morphology with a homogeneous expression of CD34, CD146, CD105, CD73 and CD90. ASC from the visceral depot secreted the highest levels of IL-6, MCP-1 and G-CSF (p = 0.0278). Interestingly, preperitoneal ASC under lipid accumulation stimulus showed the lowest levels of all the secreted cytokines, except for adiponectin that was enhanced (p = 0.0278). CONCLUSIONS: ASC from preperitoneal adipose tissue revealed the less pro-inflammatory properties, although it is an internal adipose depot. Conversely, ASC from visceral adipose tissue are the most pro-inflammatory. Therefore, ASC from subcutaneous, visceral and preperitoneal adipose depots could differentially contribute to the chronic inflammatory scenario of obesity.

Skipping of exon 27 in C3 gene compromises TED domain and results in complete human C3 deficiency.

Primary deficiency of complement C3 is rare and usually associated with increased susceptibility to bacterial infections. In this work, we investigated the molecular basis of complete C3 deficiency in a Brazilian 9-year old female patient with a family history of consanguinity. Hemolytic assays revealed complete lack of complement-mediated hemolytic activity in the patient's serum. While levels of the complement regulatory proteins Factor I, Factor H and Factor B were normal in the patient's and family members' sera, complement C3 levels were undetectable in the patient's serum and were reduced by at least 50% in the sera of the patient's parents and brother. Additionally, no C3 could be observed in the patient's plasma and cell culture supernatants by Western blot. We also observed that patient's skin fibroblasts stimulated with Escherichia coli LPS were unable to secrete C3, which might be accumulated within the cells before being intracellularly degraded. Sequencing analysis of the patient's C3 cDNA revealed a genetic mutation responsible for the complete skipping of exon 27, resulting in the loss of 99 nucleotides (3450-3549) located in the TED domain. Sequencing of the intronic region between the exons 26 and 27 of the C3 gene (nucleotides 6690313-6690961) showed a nucleotide exchange (T→C) at position 6690626 located in a splicing donor site, resulting in the complete skipping of exon 27 in the C3 mRNA.

Effects of centrifugation on cell composition and viability of aspirated adipose tissue processed for transplantation.

BACKGROUND: Centrifugation is one of the preferred methods of fat processing. Although it has been promoted for nearly three decades to separate adipose tissue components before grafting, there remain many controversies regarding the results obtained with centrifuged adipose tissue. OBJECTIVES: The authors demonstrate the effects of centrifugation on the cellular components of aspirated fat. METHODS: Fat harvested from the lower abdomen of 10 female patients undergoing liposuction was divided in two equal parts, then processed by decantation or centrifugation and sent to the laboratory. Each processed lipoaspirate was analyzed histologically after hematoxylin and periodic acid-Schiff staining for the presence of intact adipocytes. It was then cultured and analyzed by multicolor flow cytometry for identification of adipose-derived mesenchymal stem cells. RESULTS: The middle layer of the centrifuged lipoaspirate, which is used by many surgeons, showed a great majority of altered adipocytes and very few mesenchymal stem cells in comparison with the decanted sample, which maintained the integrity of the adipocytes and showed a greater number of mesenchymal stem cells. The pellet observed as a fourth layer at the bottom of the centrifuged lipoaspirate showed the greatest concentration of endothelial cells and mesenchymal stem cells, which play a crucial role in the angiogenic and adipogenic effect of the grafted tissue. CONCLUSIONS: If centrifuged lipoaspirate is used, the pellet (rich in adipose-derived mesenchymal stem cells) and the middle layer should be employed to increase fat graft survival.

Adipose tissue of control and ex-obese patients exhibit differences in blood vessel content and resident mesenchymal stem cell population.

BACKGROUND: The normal function of white adipose tissue is disturbed in obesity. After weight loss that follows bariatric surgery, ex-obese patients undergo plastic surgery to remove residual tissues and it is not known whether their adipose tissue returns to its original state. The aim of this study was to compare the white adipose tissue composition of ex-obese with control patients with regard to blood vessels and resident mesenchymal stem cells (MSC). METHODS: Quantification of blood vessels was performed on histological sections of adipose tissue stained with hematoxylin and eosin and for von Willebrand antigen. MSC were induced to the adipogenic and osteogenic lineages by specific inductive culture media. Expression of PPARgamma2 was analyzed by reverse transcription polymerase chain reaction. RESULTS: Ex-obese adipose tissue showed a higher number (p = 0.0286) of small (107.3 +/- 22.0) and large (22.5 +/- 6.4) blood vessels, when compared to control patients (42.0 +/- 24.4 and 7.2 +/- 2.2, respectively) and they also occupied a larger area (control versus ex-obese, p = 0.0286). Adipose tissue MSC from both groups of patients expressed PPARgamma2 and were equally able to differentiate to the osteogenic lineage, but ex-obese MSC showed a higher adipogenic potential when induced in vitro (p < 0.05). CONCLUSIONS: The higher number of adipose tissue blood vessels in ex-obese patients explains the excessive bleeding observed during their plastic surgery. The presence of more committed cells to the adipogenic lineage may favor the easy weight regain that occurs in ex-obese patients. These results show that, after extensive weight loss, adipose tissue cell composition was not totally restored.