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In JoVE (1)

Other Publications (19)

Articles by Paulette Conget in JoVE

 JoVE Clinical and Translational Medicine

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

1Instituto de Ciencias, Facultad de Medicina, Clínica Alemana Universidad del Desarrollo

JoVE 50050

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Other articles by Paulette Conget on PubMed

Validation in Mesenchymal Progenitor Cells of a Mutation-independent Ex Vivo Approach to Gene Therapy for Osteogenesis Imperfecta

Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.

Gp130 Activation by Soluble Interleukin-6 Receptor/interleukin-6 Enhances Osteoblastic Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.

Human Cord Blood-derived Mesenchymal Stem Cells Home and Survive in the Marrow of Immunodeficient Mice After Systemic Infusion

Bone marrow is the residence site of mesenchymal stem cells (MSC), which upon commitment and maturation develop into several mesenchymal phenotypes. Recently, we have described the presence of MSC in human cord blood (cbMSC) and informed that their properties are the same as those for MSC obtained from adult bone marrow. In this study we have investigated the capability of transplanted cbMSC to home and survive in the marrow of unconditioned nude mice. cbMSC utilized for transplantation studies were characterized by morphology, differentiation potential, and immunophenotype. After transplantation by systemic infusion, human DNA (as detected by PCR amplification of human-specific beta-globin gene) was detected in the marrow of recipients as well as in ex vivo-expanded stromal cells prepared from the marrow of transplanted animals. These results demonstrate homing and survival of cbMSC into the recipient marrow and also suggest a mesenchymal-orientated fate of engrafted cells, because human DNA was also detected in cells of other recipient tissues, like cardiac muscle, teeth, and spleen.

Subcellular Distribution and Mitogenic Effect of Basic Fibroblast Growth Factor in Mesenchymal Uncommitted Stem Cells

Uncommitted mesenchymal stem cells (MSC), upon commitment and differentiation give rise to several mature mesenchymal lineages. Although the involvement of specific growth factors, including FGF2, in the development of committed MSC is known, the effect of FGF2 on uncommitted progenitors remains unclear. We have analyzed on a comparative basis, the subcellular distribution and mitogenic effect of FGF2 in committed and uncommitted MSC prepared from human bone marrow. Indirect immunofluorescence studies showed strong nuclear FGF2 staining in both progenitors; however, cytoplasmic staining was only detected in committed cells. Western blot analysis revealed the presence of 22.5 and 21-22 kDa forms of FGF2 in the nucleus of both progenitors; however, their relative content was higher in uncommitted than in committed cells. Exogenous FGF2 stimulated proliferation and sustained quiescence in committed and uncommitted cells, respectively. These results show that both type of progenitors, apart from morphological and proliferative differences, display specific patterns of response to FGF2.

Dynamic of Distribution of Human Bone Marrow-derived Mesenchymal Stem Cells After Transplantation into Adult Unconditioned Mice

The use of mesenchymal stem cells (MSC) for cell therapy relies on their capacity to engraft and survive long-term in the appropriate target tissue(s). Animal models have demonstrated that the syngeneic or xenogeneic transplantation of MSC results in donor engraftment into the bone marrow and other tissues of conditioned recipients. However, there are no reliable data showing the fate of human MSC infused into conditioned or unconditioned adult recipients.

Basic Science of Epidermolysis Bullosa and Diagnostic and Molecular Characterization: Proceedings of the IInd International Symposium on Epidermolysis Bullosa, Santiago, Chile, 2005

Neuropotency of Human Mesenchymal Stem Cell Cultures: Clonal Studies Reveal the Contribution of Cell Plasticity and Cell Contamination

Various studies have shown neuropotency of bone marrow-derived human mesenchymal stem cells (hMSC) based on the appearance of cells with neural phenotype before or after neural induction protocols. However, to date, it is unclear which mechanisms account for this observation. We hypothesized that neural phenotypes observed in hMSC cultures can be because of both intrinsic cell plasticity and contamination by cells of neural origin. Therefore, we characterized 38 clones from hMSC cultures by assessing their adipogenic/osteogenic potential with specific mesenchymal differentiation protocols, and their molecular neural phenotype by RT-PCR analysis before and after exposure to a defined neural stem cell (NSC) medium for 8 days (neural protocol). We found 33 clones with mesenchymal potential and 15 of them also showed a neural phenotype. As neural phenotypes were maintained during the neural protocol, this suggested neural cell plasticity in 39% of all clones through pluripotency. Importantly, we were able to induce neural phenotypes in 11 of mesenchymal clones applying the neural protocol, demonstrating neural cell plasticity in 29% of all clones through the mechanism of transdifferentiation. Finally, 2 of 5 nonmesenchymal clones (5% of all clones) displayed a neural phenotype indicating neural cell contamination of hMSC cultures. In conclusion, we found 2 different ways of neuropotency of hMSC cultures: cell plasticity and cell contamination.

Systemic Administration of Multipotent Mesenchymal Stromal Cells Reverts Hyperglycemia and Prevents Nephropathy in Type 1 Diabetic Mice

Multipotent mesenchymal stromal cells (MSCs), often labeled mesenchymal stem cells, contribute to tissue regeneration in injured bone and cartilage, as well as in the infarcted heart, brain, and kidney. We hypothesize that MSCs might also contribute to pancreas and kidney regeneration in diabetic individuals. Therefore, in streptozotocin (STZ)-induced type 1 diabetes C57BL/6 mice, we tested whether a single intravenous dose of MSCs led to recovery of pancreatic and renal function and structure. When hyperglycemia, glycosuria, massive beta-pancreatic islets destruction, and mild albuminuria were evident (but still without renal histopathologic changes), mice were randomly separated in 2 groups: 1 received 0.5 x 10(6) MSCs that have been ex vivo expanded (and characterized according to their mesenchymal differentiation potential), and the other group received the vehicle. Within a week, only MSC-treated diabetic mice exhibited significant reduction in their blood glucose levels, reaching nearly euglycemic values a month later. Reversion of hyperglycemia and glycosuria remained for 2 months at least. An increase in morphologically normal beta-pancreatic islets was observed only in MSC-treated diabetic mice. Furthermore, in those animals albuminuria was reduced and glomeruli were histologically normal. On the other side, untreated diabetic mice presented glomerular hyalinosis and mesangial expansion. Thus, MSC administration resulted in beta-pancreatic islets regeneration and prevented renal damage in diabetic animals. Our preclinical results suggest bone marrow-derived MSC transplantation as a cell therapy strategy to treat type 1 diabetes and prevent diabetic nephropathy, its main complication.

Endovenous Administration of Bone-marrow-derived Multipotent Mesenchymal Stromal Cells Prevents Renal Failure in Diabetic Mice

Twenty-five to 40% of diabetic patients develop diabetic nephropathy, a clinical syndrome that comprises renal failure and increased risk of cardiovascular disease. It represents the major cause of chronic kidney disease and is associated with premature morbimortality of diabetic patients. Multipotent mesenchymal stromal cells (MSC) contribute to the regeneration of several organs, including acutely injured kidney. We sought to evaluate if MSC protect kidney function and structure when endovenously administered to mice with severe diabetes. A month after nonimmunologic diabetes induction by streptozotocin injection, C57BL/6 mice presented hyperglycemia, glycosuria, hypoinsulinemia, massive beta-pancreatic islet destruction, low albuminuria, but not renal histopathologic changes (DM mice). At this stage, one group of animals received the vehicle (untreated) and other group received 2 doses of 0.5 x 10(6) MSC/each (MSC-treated). Untreated DM mice gradually increased urinary albumin excretion and 4 months after diabetes onset, they reached values 15 times higher than normal animals. In contrast, MSC-treated DM mice maintained basal levels of albuminuria. Untreated DM mice had marked glomerular and tubular histopathologic changes (sclerosis, mesangial expansion, tubular dilatation, proteins cylinders, podocytes lost). However, MSC-treated mice showed only slight tubular dilatation. Observed renoprotection was not associated with an improvement in endocrine pancreas function in this animal model, because MSC-treated DM mice remained hyperglycemic and hypoinsulinemic, and maintained few remnant beta-pancreatic islets throughout the study period. To study MSC biodistribution, cells were isolated from isogenic mice that constitutively express GFP (MSC(GFP)) and endovenously administered to DM mice. Although at very low levels, donor cells were found in kidney of DM mice 3 month after transplantation. Presented preclinical results support MSC administration as a cell therapy strategy to prevent chronic renal diseases secondary to diabetes.

Replenishment of Type VII Collagen and Re-epithelialization of Chronically Ulcerated Skin After Intradermal Administration of Allogeneic Mesenchymal Stromal Cells in Two Patients with Recessive Dystrophic Epidermolysis Bullosa

In animal models it has been shown that mesenchymal stromal cells (MSC) contribute to skin regeneration and accelerate wound healing. We evaluated whether allogeneic MSC administration resulted in an improvement in the skin of two patients with recessive dystrophic epidermolysis bullosa (RDEB; OMIM 226600). Patients had absent type VII collagen immunohistofluorescence and since birth had suffered severe blistering and wounds that heal with scarring. Vehicle or 0.5 x 10(6) MSC were infused intradermally in intact and chronic ulcerated sites. One week after intervention, in MSC-treated skin type VII collagen was detected along the basement membrane zone and the dermal-epidermal junction was continuous. Re-epithelialization of chronic ulcerated skin was observed only near MSC administration sites. In both patients the observed clinical benefit lasted for 4 months. Thus intradermal administration of allogeneic MSC associates with type VII collagen replenishment at the dermal-epidermal junction, prevents blistering and improves wound healing in unconditioned patients with RDEB.

Human Mesenchymal Stem Cells Efficiently Manage Oxidative Stress

The transplantation of mesenchymal stem cells (MSCs) proves to be useful to treat pathologies in which tissue damage is linked to oxidative stress (OS). The aim of our work was to evaluate whether primary human MSCs (hMSCs) can manage OS. For this, in vitro we assessed the following parameters: (1) cell viability of hMSCs exposed to increasing concentrations of reactive oxygen species (ROS; source: hydrogen peroxide), reactive nitrogen species (RNS; source: S-nitroso-N-acetylpenicillamine), or both (ROS and RNS; source: 3-morpholinosydnonimine hydrochloride); (2) intracellular level of reactive species in hMSCs exposed to ROS and RNS; (3) basal gene expression and activity of superoxide dismutases, catalase, and glutathione peroxidase of hMSCs; (4) basal level of total glutathione (GSx) of hMSCs; and (5) cell viability of GSx-depleted hMSCs exposed to ROS and/or RNS. Results showed that hMSCs have a high resistance to OS-induced death, which correlates with low levels of intracellular reactive species, constitutive expression of enzymes required to manage OS, and high levels of GSx. When hMSCs were depleted of GSx they lose their capacity to manage OS. Thus, in vitro hMSCs were able to scavenge ROS and RNS and efficiently manage OS. If this potential is maintained in vivo, hMSCs could also contribute to tissue regeneration, limiting OS-induced tissue damage.

The Antidiabetic Effect of MSCs is Not Impaired by Insulin Prophylaxis and is Not Improved by a Second Dose of Cells

Type 1 diabetes mellitus (T1D) is due to autoimmune destruction of pancreatic beta-cells. Previously, we have shown that intravenously administered bone marrow-derived multipotent mesenchymal stromal cells (MSCs) allows pancreatic islet recovery, improves insulin secretion and reverts hyperglycemia in low doses streptozotocin (STZ)-induced diabetic mice. Here we evaluate whether insulin prophylaxis and the administration of a second dose of cells affect the antidiabetic therapeutic effect of MSC transplantation. Insulitis and subsequent elimination of pancreatic beta-cells was promoted in C57BL/6 mice by the injection of 40 mg/kg/day STZ for five days. Twenty-four days later, diabetic mice were distributed into experimental groups according to if they received or not insulin and/or one or two doses of healthy donor-derived MSCs. Three and half months later: glycemia, pancreatic islets number, insulinemia, glycated hemoglobin level and glucose tolerance were determined in animals that did not received exogenous insulin for the last 1.5 months. Also, we characterized MSCs isolated from mice healthy or diabetic. The therapeutic effect of MSC transplantation was observed in diabetic mice that received or not insulin prophylaxis. Improvements were similar irrespective if they received one or two doses of cells. Compared to MSCs from healthy mice, MSCs from diabetic mice had the same proliferation and adipogenic potentials, but were less abundant, with altered immunophenotype and no osteogenic potential.Our preclinical results should be taken into account when designing phase II clinical trials aimed to evaluate MSC transplantation in patients with T1D. Cells should be isolated form healthy donor, insulin prophylaxis could be maintained and a second dose, after an elapse of two months, appears unnecessary in the medium-term.

Intravenous Administration of Multipotent Stromal Cells Prevents the Onset of Non-alcoholic Steatohepatitis in Obese Mice with Metabolic Syndrome

Metabolic syndrome is secondary to obesity and characterized by dyslipidemia, insulin resistance, and hypertension. Non-alcoholic fatty liver disease is its hepatic manifestation, whose progression-limiting step is non-alcoholic steatohepatitis (NASH). The latter is characterized by lipid accumulation, hepatocyte damage, leukocyte infiltration, and fibrosis. NASH is a prodrome to cirrhosis and hepatocellular carcinoma. Multipotent stromal cells (MSCs) have been shown to be immunomodulatory and contribute to liver regeneration in acute failure conditions. Our aim was to evaluate whether MSC administration prevents the onset of NASH in obese mice with metabolic syndrome.

Mild Hypothermia Attenuates Lung Edema and Plasma Interleukin-1β in a Rat Mechanical Ventilation-induced Lung Injury Model

Recent data suggest that deep hypothermia has protective effects on experimental induced lung injury. It is not well known if these effects persist with mild hypothermia. The authors hypothesized that mild hypothermia may attenuate lung injury and decrease local and systemic proinflammatory cytokines in a rat model of injurious mechanical ventilation (MV). Twelve Sprague-Dawley male adult rats were anesthetized, intubated, and randomly allocated to normothermia group (37°C) (NT) or mild hypothermia group (34°C) (MH). After 2 hours of deleterious MV (peak inspiratory pressure [PIP] 40 cm H(2)O, zero end-expiratory pressure [ZEEP], and inspiratory fraction of oxygen [Fio(2)] 100%), arterial blood gases, lung gravimetry, and histological study were obtained. Protein content, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were measured in plasma and bronchoalveolar lavage (BAL) fluid. Subjects that underwent MH had a significant lower wet-to-dry lung weight ratio (8.32 ± 0.28 vs. 10.8 ± 0.49, P = .01), IL-1β plasma concentration (0.6 ± 0.6 vs. 10.27 ± 2.80 pg/mL, P = .0048) and PaCO(2). There were no differences in terms of PaO(2), histological injury, or BAL protein content. In this model of injurious mechanical ventilation, subjects treated with mild hypothermia had less lung edema and lower plasma IL-1β. Some of known beneficial effects of deep hypothermia can be obtained with mild hypothermia.

Insulin is Secreted Upon Glucose Stimulation by Both Gastrointestinal Enteroendocrine K-cells and L-cells Engineered with the Preproinsulin Gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.

Novel and Recurrent COL7A1 Mutations in Chilean Patients with Dystrophic Epidermolysis Bullosa

Nosema Ceranae an Emergent Pathogen of Apis Mellifera in Chile

The microsporidian Nosema apis and Nosema ceranae have been associated with colony disorders of Apis mellifera and Apis cerana, respectively. N. apis is endemic in South America. Recently, N. ceranae has been detected in Brazil, Uruguay and Argentina. No report of its presence, distribution and prevalence in Chile is available. Here, we present a real-time PCR-based method that was able to discriminate between N. apis and N. ceranae. The dynamic range of this assay was 100 to 100,000 spores per honeybee. False-negative results were avoided due to the use of ACTIN gene as internal standard. False-positive results were obtained neither in experimentally nor in naturally contaminated samples. Using this method, we screened 240 beehives from the Chilean region where 42% of the total country honey production take places (Región del Biobío). Nosema spp. were detected in the four provinces and in 20 of the 26 communes of the region. Among the samples analysed, 49% were positive for N. ceranae. Their infection level ranged from 200 to more than 100,000 spores per honeybee. N. apis was not detected in this region. Hence, our data show that in Chile N. ceranae is an emergent pathogen that is been replacing N. apis. Also, they support that N. ceranae maybe the actual responsible for nosemosis in A. mellifera in South America.

The Antidiabetic Effect of Mesenchymal Stem Cells is Unrelated to Their Transdifferentiation Potential but to Their Capability to Restore Th1/Th2 Balance and to Modify the Pancreatic Microenvironment

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease that results from cell-mediated autoimmune destruction of insulin-producing cells. In T1DM animal models, it has been shown that the systemic administration of multipotent mesenchymal stromal cells, also referred as to mesenchymal stem cells (MSCs), results in the regeneration of pancreatic islets. Mechanisms underlying this effect are still poorly understood. Our aims were to assess whether donor MSCs (a) differentiate into pancreatic β-cells and (b) modify systemic and pancreatic pathophysiologic markers of T1DM. After the intravenous administration of 5 × 10(5) syngeneic MSCs, we observed that mice with T1DM reverted their hyperglycemia and presented no donor-derived insulin-producing cells. In contrast, 7 and 65 days post-transplantation, MSCs were engrafted into secondary lymphoid organs. This correlated with a systemic and local reduction in the abundance of autoaggressive T cells together with an increase in regulatory T cells. Additionally, in the pancreas of mice with T1DM treated with MSCs, we observed a cytokine profile shift from proinflammatory to antinflammatory. MSC transplantation did not reduce pancreatic cell apoptosis but recovered local expression and increased the circulating levels of epidermal growth factor, a pancreatic trophic factor. Therefore, the antidiabetic effect of MSCs intravenously administered is unrelated to their transdifferentiation potential but to their capability to restore the balance between Th1 and Th2 immunological responses along with the modification of the pancreatic microenvironment. Our data should be taken into account when designing clinical trials aimed to evaluate MSC transplantation in patients with T1DM since the presence of endogenous precursors seems to be critical in order to restore glycemic control.

MSC Transplantation: a Promising Therapeutic Strategy to Manage the Onset and Progression of Diabetic Nephropathy

Currently, one of the main threats to public health is diabetes mellitus. Its most detrimental complication is diabetic nephropathy (DN), a clinical syndrome associated with kidney damage and an increased risk of cardiovascular disease. Irrespective of the type of diabetes, DN follows a well-known temporal course. The earliest detectable signs are microalbuminuria and histopathological changes including extracellular matrix deposition, glomerular basement membrane thickening, glomerular and mesangial expansion. Later on macroalbuminuria appears, followed by a progressive decline in glomerular filtration rate and the loss of glomerular podocytes, tubulointerstitial fibrosis, glomerulosclerosis and arteriolar hyalinosis. Tight glycemic and hypertension controls remain the key factors for preventing or arresting the progression of DN. Nevertheless, despite considerable educational effort to control the disease, a significant number of patients not only develop DN, but also progress to chronic kidney disease. Therefore, the availability of a strategy aimed to prevent, delay or revert DN would be highly desirable. In this article, we review the pathophysiological features of DN and the therapeutic mechanisms of multipotent mesenchymal stromal cells, also referred to as mesenchymal stem cells (MSCs). The perfect match between them, together with encouraging pre-clinical data available, allow us to support the notion that MSC transplantation is a promising therapeutic strategy to manage DN onset and progression, not only because of the safety of this procedure, but mainly because of the renoprotective potential of MSCs.

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