Brazilian minipig as a large-animal model for basic research and stem cell-based tissue engineering. Characterization and in vitro differentiation of bone marrow-derived mesenchymal stem cells

Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (

IFI27 transcription is an early predictor for COVID-19 outcomes, a multi-cohort observational study

PurposeRobust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness.MethodsWe conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients.ResultsWe show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients.ConclusionThese data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus

Dental pulp-derived Stem Cells: A promising source for regenerative medicine

Submitted by Manoel Barata ([email protected]) on 2019-09-19T17:55:14Z No. of bitstreams: 1 BJSTR.MS.ID.00264.pdf: 417355 bytes, checksum: 0b8f6bf21243e017ad013b397cc0b009 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-10-11T19:55:51Z (GMT) No. of bitstreams: 1 BJSTR.MS.ID.00264.pdf: 417355 bytes, checksum: 0b8f6bf21243e017ad013b397cc0b009 (MD5)Made available in DSpace on 2019-10-11T19:55:51Z (GMT). No. of bitstreams: 1 BJSTR.MS.ID.00264.pdf: 417355 bytes, checksum: 0b8f6bf21243e017ad013b397cc0b009 (MD5) Previous issue date: 2019Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Biologia Básica de Células Tronco. Curitiba, PR, Brasil.Pontifícia Universidade Católica do Paraná. Núcleo de Tecnologia Celular. Curitiba, PR, Brasil.Regenerative medicine has the potential to replace tissues and organs damaged using stem cells (SCs) and their derivatives. Mesenchymal stromal cells (MSCs) are adult cells, multipotent and functionally defined as having self-renewal capacity, differentiation capacity in several cell lines and extensive paracrine and immunomodulatory activity. It has been described the presence of different types of MSCs populations in teeth. Dental pulp-derived SCs (DPSCs) are an important source of SC for cell therapy, can easily be collected and presents a low risk for the patient besides the great availability of material (e.g.: third molar). DPSCs have differentiation potential in multiple lineages like MSCs obtained from other tissues. The use of MSCs on a large scale in the research depends on the quality of the cells that will promote the regeneration of an injured tissue. An important aspect of standardization is the isolation method. The isolated and cultivated DPSCs present fibroblast-like morphology, plastic adherence and demonstrate the immunophenotypic profile suggested by International Society for Cellular Therapy. DPSCs derive from neural crest and have the potential to differentiate into neurons and promote the survival of neuronal cells from the injured site. The characteristics (differentiation and surface markers) presented by DPSCs are very similar with stem cells of other tissues. However, for DPSC to be used on a large scale in regenerative medicine it is necessary to standardize the isolation methodology

Genetic evaluation of mesenchymal stem cells by G-banded karyotyping in a Cell Technology Center

OBJECTIVE: To present the initial results of first three years of implementation of a genetic evaluation test for bone marrow-derived mesenchymal stem cells in a Cell Technology Center.METHODS: A retrospective study was carried out of 21 candidates for cell therapy. After the isolation of bone marrow mononuclear cells by density gradient, mesenchymal stem cells were cultivated and expanded at least until the second passage. Cytogenetic analyses were performed before and after cell expansion (62 samples) using G-banded karyotyping.RESULTS: All the samples analyzed, before and after cell expansion, had normal karyotypes, showing no clonal chromosomal changes. Signs of chromosomal instability were observed in 11 out of 21 patients (52%). From a total of 910 analyzed metaphases, five chromatid gaps, six chromatid breaks and 14 tetraploid cells were detected giving as total of 25 metaphases with chromosome damage (2.75%).CONCLUSION: The absence of clonal chromosomal aberrations in our results for G-banded karyotyping shows the maintenance of chromosomal stability of bone marrow-derived mesenchymal stem cells until the second passage; however, signs of chromosomal instability such as chromatid gaps, chromosome breaks and tetraploidy indicate that the long-term cultivation of these cells can provide an intermediate step for tumorigenesis

Influence of Adipose Tissue-Derived Stem Cells on the Burn Wound Healing Process

Burns are lesions in which the thermal energy of the causative agent transfers heat to the surface of the body, causing superficial or deep damage to the skin with protein denaturation in cells and biochemical maladjustments, which delay and disrupt the cicatricial process, increasing the chances of functional and aesthetic sequelae. This study evaluates the influence of adipose tissue-derived stem cells (ADSCs) on burn healing in terms of the size of the cicatricial space and quantified measures of collagen deposition, inflammatory infiltrate, blood vessels, and lymphatic vessels. Initially, intra-abdominal adipose tissue was resected from a single donor Wistar rat that was not part of any of the subsequent groups to obtain ADSCs by isolation and cell culture. Burns were made in the left lateral abdominal region of Wistar rats by contact with a square ceramic paper with a 484 mm2 area heated to 100°C for 30 seconds. Intradermal ADSC transplantation was performed in two stages. The first was on the same day of the burn, when 3.2×106 ADSCs were transplanted shortly after the burned region cooled, while the second stage occurred four days later with the same number of ADSCs. The progress was evaluated by immunohistochemical methods and H&E, Masson’s trichrome, Picrosirius red, and Lyve-1 immunofluorescence staining. Despite the quantitative similarity of blood vessels and the inflammatory infiltrate observed by H&E, there were statistically significant differences between the groups on the fourteenth day of evolution. The group that received ADSCs showed a reduction in the scar tissue area, increased collagen type III deposition, and a quantifiable reduction in lymphatic vessels, so we conclude that ADSCs influence the healing of total thickness burns in rats

Mesenchymal stromal cells derived from exfoliated deciduous teeth express neuronal markers before differentiation induction

Objective: This study aimed to evaluate neuronal markers in stromal cells from human exfoliated deciduous teeth (SHED) and standardize the isolation and characterization of those cells. Methodology: Healthy primary teeth were collected from children. The cells were isolated by enzymatic digestion with collagenase. By following the International Society for Cell and Gene Therapy (ISCT) guidelines, SHED were characterized by flow cytometry and differentiated into osteogenic, adipogenic, and chondrogenic lineages. Colony-forming unit-fibroblasts (CFU-F) were performed to assess these cells’ potential and efficiency. To clarify the neuronal potential of SHED, the expression of nestin and βIII-tubulin were examined by immunofluorescence and SOX1, SOX2, GFAP, and doublecortin (DCX), nestin, CD56, and CD146 by flow cytometry. Results: SHED showed mesenchymal stromal cells characteristics, such as adhesion to plastic, positive immunophenotypic profile for CD29, CD44, CD73, CD90, CD105, and CD166 markers, reduced expression for CD14, CD19, CD34, CD45, HLA-DR, and differentiation in three lineages confirmed by staining and gene expression for adipogenic differentiation. The average efficiency of colony formation was 16.69%. SHED expressed the neuronal markers nestin and βIII-tubulin; the fluorescent signal intensity was significantly higher in βIII-tubulin (p<0.0001) compared to nestin. Moreover, SHED expressed DCX, GFAP, nestin, SOX1, SOX2, CD56, CD146, and CD271. Therefore, SHED had a potential for neuronal lineage even without induction with culture medium and specific factors. Conclusion: SHEDs may be a new therapeutic strategy for regenerating and repairing neuronal cells and tissues

Natural killer cell assessment in peripheral circulation and bronchoalveolar lavage fluid of patients with severe sepsis: a case control study

Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-γ). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis

Natural killer cell assessment in peripheral circulation and bronchoalveolar lavage fluid of patients with severe sepsis: a case control study

Sepsis is a complex systemic inflammatory syndrome, the most common cause of which is attributed to systemic underlying bacterial infection. The complete mechanisms of the dynamic pro- and anti-inflammatory processes underlying the pathophysiology of sepsis remain poorly understood. Natural killer (NK) cells play a crucial role in the pathophysiology of sepsis, leading to exaggerated inflammation due their rapid response and production of pro-inflammatory cytokines such as interferon gamma (IFN-gamma). Several studies have already shown that NK cells undergo lymphopenia in the peripheral blood of patients with sepsis. However, our understanding of the mechanisms behind its cellular trafficking and its role in disease development is restricted to studies in animal models. In this study, we aimed to compare the human NK cell subset (CD56(bright or dim)) levels in the peripheral blood and bronchoalveolar lavage (BAL) fluid of sepsis patients. We conducted a case-control study with a sample size consisting of 10 control patients and 23 sepsis patients enrolled at the Hospital Cajuru (Curitiba/PR, Brazil) from 2013 to 2015. Although we were able to confirm previous observations of peripheral blood lymphopenia, no significant differences were detected in NK cell levels in the BAL fluid of these patients. Overall, these findings strengthened the evidence that peripheral blood lymphopenia is likely to be associated with cell death as a consequence of sepsis183National Health and Medical Research Council of Australia; National Breast Cancer Foundation (NBCF) Fellowship; Cancer Cure Australia Priority-Driven Young Investigator Project Grant; National Breast Cancer Foundatio