Ricardo Cunha

Controversies About the Chromosomal Stability of Cultivated Mesenchymal Stem Cells: Their Clinical Use is it Safe?

The usefulness of adult stem cells in research and therapeutic applications highly relies on their genomic integrity and stability. Many laboratories including ours have addressed this concern using methods such as karyotyping, Qbanding, fluorescent in situ hybridization, array CGH, flow cytometry and Pap test to evaluate number and structure of chromosomes and cellular phenotype. This review attempts to summarize the findings reported so far for the studies on chromosomal aberrations in adult stem cells and warrant to perform certain basic tests before transplantation to avoid any adverse reactions, which will thus aid in better therapeutic output after cellular transplantation in the treatment of various diseases.

Effect of exercise associated with stem cell transplantation on ventricular function in rats after acute myocardial infarction

OBJECTIVE To analyze the functional and anatomical-pathological effect of transplantation of bone marrow mononuclear cells associated to aquatic physical activity after myocardial infarction in rats. METHODS Twenty-one rats were induced by myocardial infarction, through left coronary artery ligation. After a week, the animals were subjected to echocardiography for evaluation of left ventricle ejection fraction (LVEF, %) and dyastolic and end systolic volume of the left ventricle (EDV, ESV, ml), randomized and the transplantation of mononuclear stem cells. The animals were divided into four groups: sedentary group without cells (n=5), sedentary with cells (n=5), trained without cells (n=5) and trained with cells (n=6). The physical training was started 30 days after infarction and held in swimming during 30 days. At the beginning and at the end of the physical training protocol were held assay of lactate. The animals have been subjected to new echocardiography after 60 days of myocardial infarction. RESULTS Two months after the transplant, were observed decrease in FE in the control group (35.2 to 23.54 P=0.022) and addition of LVEF and stabilization of ventricular remodeling in the group trained with cells (29.85 to 33.43% P=0.062 and 0.71 to 0.73 ml, P=0.776, respectively). Identified the reduction of collagen fibers, myocardial fibrosis regions in the group trained with and without cells. CONCLUSION The group trained with cells improves ventricular function compared to the control group, suggesting the benefit of associated cell therapy will physical activity.

Pap Test as the First Step in Screening Genetic Stability in Cell-BasedTherapy

The possibility of cell modifications compromises the safety of stem cell therapy under standardized conditions. The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Methods: Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou (Pap) staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. Results: The Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. Conclusions: These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs as well for other adherent stem cells.

Acellular Human Amniotic Membrane Scaffold Loaded with Nanoparticles Containing 15d-PGJ2: A New System Local Anti-Inflammatory Treatment of Eye Diseases

The pathogenesis of chronic inflammatory eye diseases is multifactorial and includes factors as tissue injuries, metabolic disorder and autoimmune diseases. The 15-deoxy-Δ12, 14-PG J2 is known for its anti-inflammatory, antioxidant and immunmodulatory properties. In vivo adhesions between cells and the extracellular matrix play a crucial role in cell differentiation, proliferation, and migration as well as tissue remodeling. Here, we present a simple method to incorporate 15d-PGJ2 nanoparticles in acellular human amniotic membrane (HAM) scaffold, as potential local anti-inflammatory delivery system. After completely removing the cells on the amniotic membrane with a sodium dodecyl sulphate and mechanical approach, we seeded Vero cells incorporate 15d-PGJ2 nanoparticles on it. The morphology of the Vero cells and nanoparticles was observed by scanning electron microscopy (SEM). The cells cultivated observed by scanning electron microscopy (SEM) presented the incorporation of the nanoparticles smooth surface and spherical shape. Our results indicate that the HAM may be an ideal candidate as a nanoparticule-matrix adhesion substrate to study a new system for local anti-inflammatory therapy.

Human Adipose-Derived Mesenchymal Stem Cells Cryopreservation and Thawing Decrease α4-Integrin Expression

Aim. The effects of cryopreservation on adipose tissue-derived mesenchymal stem cells are not clearly documented, as there is a growing body of evidence about the importance of adipose-derived mesenchymal stem cells for regenerative therapies. The aim of this study was to analyze human adipose tissue-derived mesenchymal stem cells phenotypic expression (CD34, CD45, CD73, CD90, CD105, and CD49d), colony forming unit ability, viability, and differentiation potential before and after cryopreservation. Materials and Methods. 12 samples of the adipose tissue were collected from a healthy donor using the liposuction technique. The cell isolation was performed by enzymatic digestion and then the cells were cultured up to passage 2. Before and after cryopreservation the immunophenotype, cellular viability analysis by flow cytometer, colony forming units ability, differentiation potential into adipocytes and osteoblasts as demonstrated by Oil Red O and Alizarin Red staining, respectively. Results. The immunophenotypic markers expression was largely preserved, and their multipotency was maintained. However, after cryopreservation, the cells decreased α4-integrin expression (CD49d), cell viability, and number of colony forming units. Conclusions. These findings suggest that ADMSC transplanted after cryopreservation might compromise the retention of transplanted cells in the host tissue. Therefore, further studies are warranted to standardize protocols related to cryopreservation to attain full benefits of stem cell therapy.

Cardiac Analysis of Autologous Transplantation of Cocultured Skeletal Myoblasts and Mesenchymal Cells in a Rat Model Doxorubicin-Induced Cardiotoxicity: Histopathological and Functional Studies

Objective: Investigate the functional and histopathological effects of combined transplantation of mesenchymal stem cells (MSCs) and skeletal myoblasts (SM) in rats submitted to doxorubicin (DOX) induced cardiomyopathy. Methods: Myocardiopathy was induced through intraperitoneal applications of 3.75 mg/kg/day DOX once a week for 4 weeks in all experimental animals. Echocardiography examination was conducted to evaluate heart function. Myocardial cell apoptosis was examined morphologically by optical microscopy. The expression of both, von Willebrand factor and MyoD proteins was analysed using immunohistochemical staining. Results: Our results showed that all the experimental animals developed cardio-toxicity and exhibited decrease in heart function 4 weeks after the administration of DOX (P<0.05). The ventricular function significantly improved in rats that received sub-epicardial injection of co-culture of SM and MSC (CO group) when compared to rats that received only saline sub-epicardial injection (CG group) (P<0.05). Conclusion: The results of this study provide evidences that the myocardial co-culture transplantation of SM and MSCs contributed to the treatment the DOX-induced cardiotoxicity.

Myocardial regeneration after implantation of porcine small intestinal submucosa in the left ventricle

Introduction Most cardiomyocytes do not regenerate after myocardial infarction. Porcine small intestinal submucosa has been shown to be effective in tissue repair. Objective To evaluate myocardial tissue regeneration and functional effects of SIS implantation in pigs after left ventriculotomy. Methods Fifteen pigs were assigned to two groups: porcine small intestinal submucosa (SIS) (N=10) and control (N=5). The SIS group underwent a mini sternotomy, left ventriculotomy and placement of a SIS patch. The control group underwent a sham procedure. Echocardiography was performed before and 60 days after the surgical procedure. Histological analysis was performed with hematoxylin-eosin stain and markers for actin 1A4, anti sarcomeric actin, connexin43 and factor VIII. Results Weight gain was similar in both groups. Echocardiography analysis revealed no difference between groups regarding end diastolic and systolic diameters and left ventricular ejection fraction, both pre (P=0.118, P=0.313, P=0.944) and post procedure (P=0.333, P=0.522, P=0.628). Both groups showed an increase in end diastolic (P<0,001 for both) and systolic diameter 60 days after surgery (P=0.005, SIS group and P=0.004, control group). New cardiomyocytes, blood vessels and inflammatory reactions were histologically identified in the SIS group. Conclusion SIS implantation in pigs after left ventriculotomy was associated with angiomuscular regeneration and no damage in cardiac function.

The Effects of Total Thyroidectomy on Cardiac Function in Old Rats using Echocardiographic Measures

Objectives: Doppler Echocardiography has been shown to reflect thyroid hormone action in primary thyroid dysfunction. The aim of the present study was to evaluate the cardiac function after total thyroidectomy in old rats using echocardiographic measures. Methods: Female Wistar rats aged 24 months old (referred to as adult) were divided into 2 groups with 15 animals each: Euthyroid (Eut) and Hypothyroid (Hypo) rats. Hypothyroidism was induced by total thyroidectomy. The plasmatic levels of T3, T4, TSH and free ionic calcium were measured. Echocardiographic analysis was performed using the following parameters: left ventricular end-diastolic (LVED) left ventricular end-systolic (LVES), ejection fraction (EF) and Tei index. Results: In both groups, the LVES ranged from 0.2 to 0.5 p <0.048 and the EF from 50.3 to 45.2 p<0.030.There was not statistically significant difference in HR, LVED and Tei-index values. It was observed decrease in T3 levels (72.0 to 30.4 μg/dl, p<0.001) and T4 levels (3.1 to 0.4 μg/dl, p<0.001) and increase in TSH levels (1.6 to 40.7 mU/mol, p<0.001) in Hypo group. The free ionic calcium levels increased from 0.1 to 0.9 mmol/L (p<0.001) in Hypo group. Conclusion: It was identified a reduction of cardiac function in old animals with hypothyroidism using Doppler- Echocardiography measures. These results indicated that hypothyroidism is associated with impairment in cardiac function in old patients and that Doppler echocardiography, is a useful and noninvasive tool for the diagnosis of cardiac dysfunction induced by this thyroid disorder.

15d-PGJ2-loaded in nanoparticles associated with decellurazied human amniotic membrane scaffold: A potential anti-inflammatory delivery system

Chronic inflammatory diseases are disorders multifactorial growing on elderly and diabetic populations. 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) acts in protective role against oxidative stress and inflammation both in vivo and in vitro. Here, we present a simple method to incorporate 15d-PGJ2 nanoparticles in acellular human amniotic membrane scaffold, a potential local anti-inflammatory delivery system. After completely removing the cells on the amniotic membrane with a sodium dodecyl sulphate and mechanical approach, we seeded Vero cells incorporated with 15d-PGJ2 nanoparticles. The morphology of the Vero cells and nanoparticles was observed by scanning electron microscopy (SEM) and phase contrast microscopy. The cells cultivated presented the incorporation of the nanoparticles, smooth surface and spherical shape. Within this study it was shown that amniotic membrane can be used to incorporate cells and anti-inflammatory loaded nanoparticles for prevention and treatment of various diseases. Introduction The incidence of people with chronic inflammatory diseases has been increasing over the last three decades, threatening human health. A number of factors are recognized as causes of the pathogenesis as for example the autoimmune diseases, metabolic disorders and chronic respiratory disease [1]. The 15-Deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is a natural ligand of peroxisome proliferator activated receptor γ(PPARγ). This molecule has anti-inflammatory property and is involved in a variety of physiological and pathological processes, including rheumatoid arthritis, myocardial infarction, neural damage, and tumorigenesis [2]. The nanomaterials could provide a revolution in technology that will soon impact the diseases treatment methods through new nanoparticles delivery systems [3]. The development of new strategies for regenerative medicine is one of the most active research in the areas of nanotechnology[4]. Current advances in biotechnology uses acellular scaffolds to regenerate tissues, this is an approach highly beneficial in terms of biocompatibility and biofunctionality [5]. The decellularized human amniotic membrane is the commonest sources for scaffolds used in tissue regeneration because of it good biocompatibility and biodegradability [6]. In this study, we aimed to incorporate 15-deoxy-Δ12,14-PG J2 nanoparticles in acellular human amniotic membrane scaffold as a potential local anti-inflammatory delivery system. Methods Preparation of acellular human amniotic scaffold The study was approved by the Hospital Pequeno Príncipe Ethical Committee for the usage of biological material for research purposes approved under article number 0948-11. All materials were used in compliance with ethical guidelines by the Brazilian National Health Council. Fresh human amniotic membrane (HAM) was obtained after caesarian deliveries. Maternal donors provided informed consent and were serologically negative for HIV, hepatitis B, hepatitis C, and syphilis. Briefly, blood clots were immediately cleaned off the placenta after surgery with phosphate buffered saline (PBS) solution containing 100 U/mL penicillin and 100 mg/mL streptomycin. The acellular human amniotic scaffold (AHAS) was prepared as Correspondence to: Julio Cesar Francisco, Cell Therapy and Biotechnology in Regenerative Medicine Research Group, Pelé Pequeno Príncipe Institute, Paraná, Brazil, E-mail: julio.apfr@gmail.com Received: March 10, 2016; Accepted: April 05, 2016; Published: April 11, 2016 Francisco JC (2016) 15d-PGJ2-loaded in nanoparticles associated with decellurazied human amniotic membrane scaffold: A potential anti-inflammatory delivery system Volume 2(2): 111-113 Front Nanosci Nanotech, 2016 doi: 10.15761/FNN.1000118 described by Riau et al., 2010 [7]. Part of the human HAM was then deprived of amniotic epithelial cells to obtain AHAS by sodium dodecyl sulphate in PBS, and incubated with shaking rate of 100 rpm at 37°C for 24 h. Finally, the prepared AHAS was rewashed 3 times with PBS. Nanoencapsulation of 15d-PGJ2 in poly(D,L-lactide-co-glycolide) (PLGA) nanocapsules (15d-PGJ2-NC). The 15d-PGJ2-NC were prepared by the nanoprecipitation method, as described by Fessi et al., 1989, and supplied by Dr. Napimoga from Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center [8,9]. Culture and seeding of VERO cells with 15d-PGJ2-NC Vero cells (ATCC® CCL-81TM) were cultured for 7 days in DMEM (Dulbecco’s modified Eagle medium, Invitrogen) supplemented with 10% FBS (fetal bovine serum, Invitrogen) and 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C with 5% CO2. The culture medium was changed every 2 days. The Vero cells were seeded on plastic plate wells over AHAS and with 15d-PGJ2-NC in concentration of 10 μg/ mL for 24h in order to cells incorporate the nanoparticles, after the incubation time the plate was washed with PBS. Phase contrast images were obtained with Axio Vert.A1 (Zeiss). Scanning electron microscopy (SEM) The morphology and structure of the cells with 15d-PGJ2-NC cultivated in AHAS were examined in a JEOL 1200EX II microscope (Jeol ltda, Akishima) operating at 80 kV. In order to perform the SEM analysis, material was fixed on top coverslip, dried, mounted on a stub for SEM, fixed in 2.5% (v/v) glutaraldehyde (Sigma-Aldrich) in PBS and post-fixed with 1% (v/v) and 0.1 M sodium cacodylate trihydrate (Sigma-Aldrich). Results SEM morphology Nanoparticles characteristics of 15d-PGJ2 in the Vero cells cultivated on acellular amniotic membrane were revealed by SEM analysis. Figure 1 shows the vero cells cultivated on AHAS (a), Vero cells incubated with 15d-PGJ2 10 μg/mL (b). Vero cell cultivated on AHAS and incubated with 15d-PGJ2 10 μg/mL at 6.500x (c) and 13.000x (d). The presence of 15d-PGJ2 in the surface the amniotic membrane can be noticed. At 15d-PGJ2 concentration (10 μg/mL), a more uniform covering of the cell is observed. Discussion In recent decades, the nanotechnology has progressed rapidly. One of the most important factors in regenerative medicine is the preparation of a scaffold. Acellular amniotic membrane human scaffolds have recently become the focus of interest mainly due to the possible beneficial and applications in regenerative medicine [10]. So far, various decellularization methods have been suggested to develop extracellular matrix derived scaffolds. Tissue engineering using acellular scaffods has introduced a new field of repair in the treatment of wounds tissues or diseases [11]. HAM is an appropriate substitute for reconstruction of various organs and tissues due to its availability, low cost and low risk of viral disease transmission and immunologic rejection [12]. The overall aim of this study was to develop a type of surface covered by nanoparticles and cells with tissue engineering applications in the form of a 3D scaffold. The use of chemical approach for decellularization shown in SEM completely removal of cells while leaving the matrix intact, unlike in other studies[13]. An intact extracellular matrix is essential for recellularization, because the extra cellular matrix contains components necessary for cell attachment, proliferation, and ultimately tissue remodeling [14]. In this study, we demonstrated the recellularization efficiency with the cultivation of VERO cells in AHAS provide an adequate in vitro microenvironment for proliferation and incorporation 15d-PGJ2 nanoparticles in acellular scaffold. Conflict of interest None declared. Conclusion 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is one of the potent ligand of peroxisome proliferator-activated receptor γ (PPARγ), suppresses proliferation and induces intracellular apoptosis in different events in excessive inflammation. This study showed that AHAS could be used as a biomaterial and be applied as a scaffold for regenerative medicine and nanoparticle delivery.

Bone Marrow Mononuclear Stem Cell Transplant in Acute and Chronic Arterial Insufficiency in Rabbits

Introduction: There is a high incidence of Peripheral Obstructive Arterial Disease (POAD) in patients with atherosclerosis. In more complex cases for which surgical revascularization is not possible, the only option involves clinical treatment that in the majority of cases evolves to amputation of the limb. Transplant of mononuclear stem cells from bone marrow has presented favorable results in chronic obstructions. Objective: To perform a functional analysis of the effect of bone marrow mononuclear stem cell transplant on acute arterial occlusion immediately and 48 hours after occlusion, comparing between groups and with controls. Materials and methods: Twenty New Zealand rabbits were anesthetized with ketamine and xylazine (50 mg/ kg) and underwent occlusion of the right iliac artery. Those animals that presented absence of arterial flow after ligation were included in the study. These animals were then randomized and divided into four groups: Group 1(n=5) control of acute ischemia group–injection of saline solution, Group 2(n=5) control of chronic ischemia–injection of saline solution 48hrs after occlusion. Group 3(n=5) transplant of stem cell in acute ischemia group, and Group 4(n=5) transplant of stem cells in the chronic ischemia group, 48hrs after occlusion. The animals were evaluated by the Tarlov’s movement scale, degree of tissue ischemia, and degree of modified ischemia on the seventh, fourteenth and thirtieth day after arterial occlusion. This evaluation was performed in a blind and randomized fashion by two different observers. The animals underwent another vascular Doppler exam on the thirtieth day after arterial occlusion. Results: All animals were considered homogeneous in the pre-transplant period. No statistical differences were identified between groups G1 and G3 (p=109) with respect to Tarlov scale. Regarding the intergroup analysis, a clinical improvement was observed in Group 4 when compared to Groups 1, 2, and 3, p=0.003, p=0.0025, and p=0.055 respectively, on the thirtieth day after occlusion. No significant difference was observed for the degree of ischemia and modified ischemia parameters after transplant. Conclusion: Clinical improvement in the chronic ischemia group receiving cell transplant of mononuclear stem cells was observed in comparison to the control group and in relationship to the acute ischemia group, suggesting a functional improvement in the affected limb.