Mahmoud M. Gabr

Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice.

Harvesting, expansion, and directed differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) could provide an autologous source of surrogate β-cells that would alleviate the limitations of availability and/or allogenic rejection following pancreatic or islet transplantation. Bone marrow cells were obtained from three adult type 2 diabetic volunteers and three nondiabetic donors. After 3 days in culture, adherent MSCs were expanded for two passages. At passage 3, differentiation was carried out in a three-staged procedure. Cells were cultured in a glucose-rich medium containing several activation and growth factors. Cells were evaluated in vitro by flow cytometry, immunolabeling, RT-PCR, and human insulin and c-peptide release in responses to increasing glucose concentrations. One thousand cell clusters were inserted under the renal capsule of diabetic nude mice followed by monitoring of their diabetic status. At the end of differentiation, ~5–10% of cells were immunofluorescent for insulin, c-peptide or glucagon; insulin, and c-peptide were coexpressed. Nanogold immunolabeling for electron microscopy demonstrated the presence of c-peptide in the rough endoplasmic reticulum. Insulin-producing cells (IPCs) expressed transcription factors and genes of pancreatic hormones similar to those expressed by pancreatic islets. There was a stepwise increase in human insulin and c-peptide release by IPCs in response to increasing glucose concentrations. Transplantation of IPCs into nude diabetic mice resulted in control of their diabetic status for 3 months. The sera of IPC-transplanted mice contained human insulin and c-peptide but negligible levels of mouse insulin. When the IPC-bearing kidneys were removed, rapid return of diabetic state was noted. BM-MSCs from diabetic and nondiabetic human subjects could be differentiated without genetic manipulation to form IPCs that, when transplanted, could maintain euglycemia in diabetic mice for 3 months. Optimization of the culture conditions are required to improve the yield of IPCs and their functional performance.

Generation of Insulin-Producing Cells from Human Bone Marrow-Derived Mesenchymal Stem Cells: Comparison of Three Differentiation Protocols

Introduction. Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs) to form insulin-producing cells (IPCs). We compared the relative efficiency of three differentiation protocols. Methods. Human bone marrow-derived MSCs (HBM-MSCs) were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol), trichostatin-A-based (two-step protocol), and β-mercaptoethanol-based (three-step protocol). At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. Results. By immunolabeling, the proportion of generated IPCs was modest (≃3%) in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. Conclusion. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells into Insulin-Producing Cells: Evidence for Further Maturation In Vivo

The aim of this study was to provide evidence for further in vivo maturation of insulin-producing cells (IPCs) derived from human bone marrow-derived mesenchymal stem cells (HBM-MSCs). HBM-MSCs were obtained from three insulin-dependent type 2 diabetic volunteers. Following expansion, cells were differentiated according to a trichostatin-A/GLP protocol. One million cells were transplanted under the renal capsule of 29 diabetic nude mice. Blood glucose, serum human insulin and c-peptide levels, and glucose tolerance curves were determined. Mice were euthanized 1, 2, 4, or 12 weeks after transplantation. IPC-bearing kidneys were immunolabeled, number of IPCs was counted, and expression of relevant genes was determined. At the end of in vitro differentiation, all pancreatic endocrine genes were expressed, albeit at very low values. The percentage of IPCs among transplanted cells was small (≤3%). Diabetic animals became euglycemic 8 ± 3 days after transplantation. Thereafter, the percentage of IPCs reached a mean of ~18% at 4 weeks. Relative gene expression of insulin, glucagon, and somatostatin showed a parallel increase. The ability of the transplanted cells to induce euglycemia was due to their further maturation in the favorable in vivo microenvironment. Elucidation of the exact mechanism(s) involved requires further investigation.

Tunica albuginea acellular matrix graft for treatment of Peyronie's disease. An experimental study in dogs

Objective: To study the value of an acellular matrix graft of the tunica albuginea for reconstruction of the penis in cases of severe Peyronies disease. Material and Methods: In nine mongrel dogs, an acellular matrix graft of the tunica albuginea was used to cover a 30×10 mm 2 tunical defect. Equal numbers of animals were sacrificed at 1, 3 and 6 months after surgery. Before death, an erection was induced by means of papaverine injection and cavernosography was performed. After death the penis was prepared for histopathological study. Results: All animals survived the surgery and none developed haematoma, wound infections or dehiscence. All dogs developed a straight, rigid erection. Cavernosography showed patent corpora cavernosa in all animals. The papaverine injection and cavernosographic results did not change over time. Inspection of the graft site and measurement of its length and width showed healing with no contracture. Histologically, the regenerated matrix appeared thicker than the neighbouring tunica albuginea in the 1‐month group; otherwise the appearance was normal. Gradual orientation of the fibrocytes, capillaries and collagen fibres was demonstrated at 1 month and was complete at 3 and 6 months. Comparison between an implanted tunica at 6 months and a control tunica from a normal dog showed no significant histological difference. Conclusion: A homologous acellular matrix graft of the tunica albuginea may be an alternative treatment for severe cases of Peyronies disease.

Purified murine islet allografts : Islet engraftment as influenced by implantation site and glucotoxicity

A UNIQUE and natural approach to immunoisolation of islet grafts was explored by investigating different immunoprivileged sites of the body: the brain, the testis, and the renal subcapsular space. For the intraabdominally placed testis, immunologic privilege has been, undoubtfully, demonstrated and attributed to locally produced factors that inhibit the immune response. However, the clinical inapplicability and the possible malignant transformation of the germ cells stand as major drawbacks of grafting the islets in such an unconventional organ site. The data about renal subcapsular space are contradictory. Whereas some investigators reported failure of islet allografts to function and survive in the renal subcapsular space, others demonstrated a remarkable success of islet allografting in this place. We investigated the engraftment, survival, and metabolic function of murine islet allografts when placed in two organ sites, the intraabdominally placed testis and the renal subcapsular space, without immunosuppression.

A newly developed silymarin nanoformulation as a potential antidiabetic agent in experimental diabetes

AIM This study aimed to develop a new stable nanoformulation of silymarin (SM) with optimum enhanced oral bioavailability and to evaluate its effect as well as mechanism of action as a superior antidiabetic agent over native SM using streptozotocin-induced diabetic rats. MATERIALS AND METHODS SM-loaded pluronic nanomicelles (SMnp) were prepared and fully characterized. Biochemical parameters were performed as well as histological, confocal and reverse-transcription polymerase chain reaction studies on pancreatic target tissues. RESULTS & CONCLUSION SMnp were found to improve significantly the antihyperglycemic, antioxidant and antihyperlipidemic properties as compared with native SM. In addition, SMnp was found to be a more efficient agent over SM in the management of diabetes and its associated complications due to its superior bioavailability in vivo, and the controlled release profile of SM. [Formula: see text].

Cell-seeded tubular acellular matrix for replacing a long circumferential urethral defect in a canine model: Is it clinically applicable?

Abstract Objective:To evaluate the feasibility of replacing a relatively long segment of the canine urethra by a tube of cell-seeded acellular collagen bladder matrix. Materials and methods: The study included 14 female mongrel dogs in which a 3-cm segment of the whole urethral circumference was excised and replaced by a tube of acellular matrix seeded with autologous urothelial cells. The acellular matrix was obtained from the excised bladder of female donor dogs that were not included in the study. Autologous cells were obtained from the study dogs by open bladder biopsy, with subsequent in vitro expansion and cultivation. Urethroplasty was performed over a 16 F urethral catheter that was kept for 4 weeks. The dogs were killed humanely (one every week for 4 weeks and then one monthly for 10 months). After stent removal, retrograde urethrography was used each month in the living dogs. If retention occurred a urethrogram was taken and then the dog was killed humanely. All grafts from dogs were harvested and sent for histopathological examination. Results: Exploration at 1, 2, 3 and 4 weeks showed progressive shrinkage in length, together with relative narrowing of the lumen. Three dogs developed retention within a week after stent removal and the other seven developed retention within 4 months. Retrograde urethrograms showed evidence of stricture and/or fistula at the graft site in all dogs. On exploration, grafts showed marked shrinkage (0.6–1.2 cm in length) with complete obliteration of their lumens. Histopathological examination showed extensive fibrosis of the matrix with no evident urothelial architecture. Conclusion: Cell-seeded acellular matrix tube is insufficient to replace a 3-cm circumferential urethral defect in dogs.

Nanoformulated natural therapeutics for management of streptozotocin-induced diabetes: potential use of curcumin nanoformulation

AIM The goal of this study was to improve curcumin (CUR) aqueous solubility and bioavailability via nanoformulation, and then study its activity and mechanism of action as an antidiabetic agent. METHODS CUR-loaded pluronic nanomicelles (CURnp) were prepared and characterized. Biochemical assessments were performed as well as histological, confocal and RTPCR studies on pancreatic target tissues. RESULTS CURnp with a diameter of 333 ± 6 nm and ζ potential of -26.1 mv were obtained. Antidiabetic action of CURnp was attributed to significant upregulation of Pdx-1 and NKx6.1 gene expression and achievement of optimum redox balance, which led to alleviation of streptozotocin-induced β-cell damage via a significant upregulation in insulin gene expression proved by RTPCR studies and by the presence of 40% insulin positive cells through confocal microscope studies on pancreatic tissue.

From Human Mesenchymal Stem Cells to Insulin-Producing Cells: Comparison between Bone Marrow- and Adipose Tissue-Derived Cells

The aim of this study is to compare human bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs), for their differentiation potentials to form insulin-producing cells. BM-MSCs were obtained during elective orthotopic surgery and AT-MSCs from fatty aspirates during elective cosmetics procedures. Following their expansion, cells were characterized by phenotyping, trilineage differentiation ability, and basal gene expression of pluripotency genes and for their metabolic characteristics. Cells were differentiated according to a Trichostatin-A based protocol. The differentiated cells were evaluated by immunocytochemistry staining for insulin and c-peptide. In addition the expression of relevant pancreatic endocrine genes was determined. The release of insulin and c-peptide in response to a glucose challenge was also quantitated. There were some differences in basal gene expression and metabolic characteristics. After differentiation the proportion of the resulting insulin-producing cells (IPCs), was comparable among both cell sources. Again, there were no differences neither in the levels of gene expression nor in the amounts of insulin and c-peptide release as a function of glucose challenge. The properties, availability, and abundance of AT-MSCs render them well-suited for applications in regenerative medicine. Conclusion. BM-MSCs and AT-MSCs are comparable regarding their differential potential to form IPCs. The availability and properties of AT-MSCs render them well-suited for applications in regenerative medicine.

Upregulation of heme oxygenase 1 (HO-1) attenuates kidney damage, oxidative stress and inflammatory reaction during renal ischemia/reperfusion injury

The current study investigated the effect of upregulation of heme oxygenase 1 (HO-1) by cobalt protoporphyrin (CoPP) on renal dysfunctions in renal ischemia/reperfusion (I/R) injury and its underlying mechanisms. 72 male Sprague Dawley rats were divided into 4 groups: sham group, ischemic group (left 45-min renal ischemia), CoPP-before group (as ischemic group with CoPP 20 mg/kg 30 min before ischemia) and CoPP-after group (as ischemic group with CoPP 20 mg/kg 20 min after ischemia). Serum creatinine, urea and TGF-β1 and markers of redox state (MDA, SOD, GSH and CAT), nitric oxide (NO), TGF-β1 and HO-1 in kidney tissues were measured. Serum creatinine and urea levels were significantly increased in ischemic group and attenuated in CoPP-treated groups (p < 0.05). Also, markers of redox state showed significant deteriorations in ischemic group which were improved significantly in CoPP-treated groups (p < 0.05). HO-1 expression in kidney tissues showed significant increase in ischemic group and showed more significant increase in CoPP-treated groups (p < 0.05). Moreover, serum and renal TGF-β1 levels were significantly increased in ischemic group and attenuated in CoPP-treated groups (p ≶ 0.05). We concluded that up-regulation of HO-1 by CoPP treatment before and after renal I/R injury improved the kidney function and morphology and this might be due to impairment of oxidative stress and inflammatory cytokines in kidney tissues.