Compensatory renal growth after unilateral or subtotal nephrectomy in the ovine fetus.

BACKGROUND: Clinical and experimental studies show that unilateral (1/2Nx) and subtotal nephrectomy (5/6Nx) in adults result in compensatory renal growth without formation of new nephrons. During nephrogenesis, the response to renal mass reduction has not been fully investigated. METHODS: Ovine fetuses underwent 1/2Nx, 5/6Nx, or sham surgery (sham) at 70 d of gestation (term: 150 d), when nephrogenesis is active. At 134 d, renal function was determined, fetuses were killed, and kidneys were further analyzed at the cellular and molecular levels. Additional fetuses subjected to 5/6Nx were killed at 80 and 90 d of gestation to investigate the kinetics of the renal compensatory process. RESULTS: At 134 d, in 1/2Nx, a significant increase in kidney weight and estimated glomerular number was observed. In 5/6Nx, the early and marked catch-up in kidney weight and estimated glomerular number was associated with a striking butterfly-like remodeling of the kidney that developed within the first 10 d following nephrectomy. In all groups, in utero glomerular filtration rates were similar. CONCLUSION: Compensatory renal growth was observed after parenchymal reduction in both models; however, the resulting compensatory growth was strikingly different. After 5/6Nx, the remnant kidney displayed a butterfly-like remodeling, and glomerular number was restored.

Laparoscopy in ureteral engineering: a feasibility study.

OBJECTIVE: We recently bioengineered a ureter substitute from a seeded scaffold implanted by open surgery in the omentum. In view of the development of laparoscopy in the treatment of benign conditions of the ureter, obtaining a ureter substitute by minimally invasive techniques would be a desirable objective. However, conflicting results about the biological impact of carbon dioxide insufflation on the microcirculation of intra-abdominal organs prompted us to investigate first whether the results obtained by open surgery, in terms of vascular supply and maturation, could be reproduced laparoscopically. MATERIALS AND METHODS: Bladder full-thickness tissue was harvested laparoscopically from three pigs for urothelial and smooth muscle cell primary cultures subsequently used to seed a small intestinal submucosa (SIS) matrix. After 2 wk, the in vitro seeded constructs were shaped around silicone drains and transferred laparoscopically into the abdomen for omental maturation. Three weeks later, the constructs were harvested for histological, immunohistochemical, and electron microscopic analysis. RESULTS: The laparoscopic procedures were performed successfully in all animals. After omental maturation, the constructs were vascularized and comprised of a well-differentiated multilayered urothelium with umbrella cells, over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. Electron microscopic analysis showed characteristics of a terminally differentiated urothelium. CONCLUSION: As shown by conventional microscopy, immunochemistry, and electron microscopy, carbon dioxide insufflation does not impact cell growth and differentiation. These findings validate the laparoscopic approach for omental maturation of ureter substitutes.

Intra renal arterial injection of autologous mesenchymal stem cells in an ovine model in the postischemic kidney.

BACKGROUND AND AIMS: Acute renal failure (ARF) remains a major healthcare problem. Although modern medical therapy has improved its outcome, the syndrome still has high mortality and morbidity rates [Xue et al.: J Am Soc Nephrol 2006;17:1135-1142]. Recently, stem cell (SC) therapies have been proposed as an alternative for the treatment of ARF on the basis of the damaged cells' replacement or enhanced recovery or regeneration. The aims of this study were to investigate the engraftment of autologous mesenchymal stem cells (MSC) injected into the renal artery in an ovine model of ischemia reperfusion injury (IRI) and to assess the consequence of the delay between injury and cell transplantation on the engraftment. MATERIAL AND METHODS: MSC were transplanted in animals submitted to IRI or in healthy animals not submitted to IRI. Sheep with IRI were grafted at two different time points after injury. Unilateral renal IRI was performed by percutaneous transluminal placement of a balloon catheter in the renal artery. MSC were isolated from bone marrow, cultured, labeled and injected into the renal artery. RESULTS: All ewes showed renal engraftment by MSC, both in tubules and glomeruli. MSC expressed tubular epithelial cell markers and podocyte phenotype. There was a significant increase of engraftment of tubules by MSC when cells were injected early after injury indicating that the delay for cell transplantation after ischemic insult should be short. CONCLUSIONS: This is the first report of intra-arterial autologous transplantation of MSC in the kidney, resulting in a successful engraftment into tubular and glomerular structures. The results strongly suggest that the optimal time window for stem cell therapy is during the early phase of the ischemic injury.

Terminal urothelium differentiation of engineered neoureter after in vivo maturation in the "omental bioreactor".

OBJECTIVE: Long ureteric defects may theoretically be repaired with the use of tissue-engineered neoureter. However, attempts to construct such a neoureter in animal models have failed because of major inflammatory response. Avoidance of such inflammation requires a well-differentiated urothelium. We investigated whether omental maturation of a seeded construct in a pig model could achieve terminal differentiation of the urothelium to allow construction of a stricture-free neoureter. MATERIAL AND METHOD: Bladder biopsies were taken to allow urothelial and smooth muscle cell cultures. These cultured cells were used to seed small intestinal submucosa (SIS) matrix. After 2 wk of cell growth, the in vitro SIS-seeded construct was shaped around a silicone drain and wrapped by the omentum to obtain neoureters. These neoureters were left in the omentum without any contact with urine, and then harvested 3 wk later for histologic and immunohistochemical studies. RESULTS: Before implantation, the in vitro constructs were composed of a mono- or bilayer of undifferentiated urothelium overlying a monolayer of smooth muscle cells. After 3 wk of omental maturation, these constructs were vascularized and comprised a terminally differentiated multilayered urothelium with umbrella cells over connective tissue and smooth muscle cells, with no evidence of fibrosis or inflammation. CONCLUSION: We obtained, for the first time, with this model of in vivo maturation in the omentum, a mature neoureter composed of a well-differentiated multilayered urothelium.

Non-cultured cell transplantation in an ovine model of non-ischemic heart failure.

OBJECTIVE: Cell therapy may be a promising alternative or adjunct to current treatment modalities for ischemic heart failure. But little is known on the impact of myogenic cell transplantation in large animal models of non-ischemic cardiomyopathy. The aim of the present study was to explore whether an ovine model of toxin-induced heart disease could benefit from non-cultured skeletal muscle cell transplantation. METHODS: Sequential intracoronary injections of doxorubicin (0.75 mg/kg) were carried out every 2 weeks until echocardiographic detection of myocardial dysfunction. Sheep were then randomly assigned to either non-cultured cell transplantation (n=8) or placebo injection (n=5). For the cell therapy group, a skeletal muscle biopsy (about 10 g) was explanted from each animal approximately 3h before grafting. After thoracotomy, 20 epicardial injections were carried out. The animals were assessed one last time before sacrifice, 2 months after the thoracotomy. Cells were tracked with cmDiI (red fluorescence) and characterized with immunohistochemistry with monoclonal antibodies to a fast skeletal isoform of myosin heavy chain. RESULTS: Two months after intramyocardial grafting, tissue Doppler imaging and conventional echocardiographic assessment of the groups showed a marked improvement in the non-cultured cell therapy group. Ejection fraction (EF) (p<0.05) as well as systolic endocardial velocities (p<0.01) improved versus the placebo group. CmDiI and skeletal myosin heavy chain expression was detected in all animals at 2 months after implantation confirming engraftment of skeletal muscle cells. CONCLUSIONS: In conclusion, our data indicate that non-cultured muscle cell transplantation is feasible and may translate into a functional benefit in an ovine model of dilated heart failure.

Development of a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for bladder tissue engineering.

OBJECTIVES: Tissue engineering is very promising in bladder reconstruction. However, one of the main problems is to limit the development of ischaemic fibrosis during tissue maturation. We describe a model using the omentum as an in vivo bioreactor for a previously seeded scaffold. METHODS: Bladder biopsies were taken from five female pigs, from which both urothelial and smooth muscle cells cultures were made. These cultured cells were used to seed a sphere-shaped small intestinal submucosa (SIS) matrix, which was transferred into the omentum after 3 wk of cell growth. The grafts were harvested 3 wk later and histologic, immunohistochemical, and functional studies were performed. RESULTS: We obtained a highly vascularized tissue-engineered construct that contracted in response to acetylcholine stimulation. The wall thickness was 4mm, on average. Histologic and immunostaining analysis of the construct confirmed the presence of a multilayer urothelium on the luminal aspect and deeper fascicles organised tissue composed of differentiated smooth muscle cells and mature fibroblasts without evidence of inflammation or necrosis. Large- and small-diameter vessels were clearly identified histologically in the tissue obtained. CONCLUSION: The omentum permitted in vivo maturation of seeded scaffolds with the development of a dense vascularisation that is anticipated to prevent fibrosis and loss of contractility. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.

Successful engraftment of cultured autologous mesenchymal stem cells in a surgically repaired soft palate defect in an adult horse.

The objective of this study was to graft autologous mesenchymal stem cells (MSCs) at the site of surgical repair of a soft palate defect in an adult horse in an attempt to improve wound healing and to investigate whether the transplanted MSCs would integrate into the soft palate structure and participate in regeneration. Bone marrow was collected from an adult horse with a full-thickness soft palate defect. The MSCs were isolated, cultured in monolayers, and labeled with 5-bromo-2-desoxymidine (BrdU) and chloromethylbenzamido-DiI-derived (cm-DiI) before transplantation. The soft palate defect was repaired by mandibular symphysiotomy, and the labeled MSCs were injected into the repaired soft palate. Postmortem examination revealed that 90% of the soft palate defect had been sutured. Staining by BrdU and cm-DiI was intense in the soft palate tissue. Labeled MSCs were detected in tissue slices from the injection sites. The cells were organized in a manner similar to that in native soft palate tissue, indicating successful engraftment.

Noncultured cell transplantation in an ovine model of right ventricular preparation.

OBJECTIVE: Beyond the first 2 months of life, pulmonary artery banding is warranted before two-stage arterial switch operation. The aim of this study was to explore whether myogenic cell transplantation could contribute to right ventricular function during pulmonary artery constriction in an ovine model. METHODS: Sixteen rams were assigned to one of the following groups: group 1, simple pulmonary artery banding (n = 5); group 2, pulmonary artery banding and cell implantation in the right ventricle (n = 7); and group 3, pulmonary artery banding and placebo injection in the right ventricle (n = 4). Hemodynamic assessment with pressure-volume loops was performed on days 0 and 60. The pulmonary artery banding and the injections were achieved through a left fourth intercostal thoracotomy. Autologous myogenic cell implantation was carried out with a noncultured cell preparation, as previously described by our group. Implanted sites were processed with monoclonal antibodies to a fast skeletal-specific isoform of myosin heavy chain (MY32). RESULTS: Skeletal myosin heavy chain expression was detected at 2 months after noncultured cell implantation in all grafted animals. Right ventricular training resulted in statistically significant increased signs of contractility in all three groups. There was no observed difference, however, between the cell therapy group and the other two groups with respect to signs of cardiac function. CONCLUSION: Successful engraftment of noncultured cells into right ventricular myocardium did not translate into a functional benefit that we could demonstrate in our ovine model. Cellular therapy thus is probably not useful to strengthen a left ventricle being retrained through pulmonary artery banding before arterial switch operation. However, cell transplantation may affect the outcome of right ventricular failure long term after atrial switch operation. Although preliminary, this investigation paves the way for further research into cellular cardiomyoplasty, right ventricular failure, and congenital heart disease.

Noncultured, autologous, skeletal muscle cells can successfully engraft into ovine myocardium.

BACKGROUND: There is compelling evidence showing that cellular cardiomyoplasty can improve cardiac function. Considering the potential benefit of using noncultured muscle cells (little time, lower cost, reduced risk of contamination), we investigated the feasibility of grafting cells obtained directly after enzymatic dissociation of skeletal muscle biopsies into ovine myocardium. We hypothesized that those noncultured muscle cells would engraft massively. METHODS AND RESULTS: Autologous, intramyocardial skeletal muscle cell implantation was performed in 8 sheep. A skeletal muscle biopsy sample ( approximately 10 g) was explanted from each animal. The sheep were left to recover for approximately 3 hours and reanesthetized when the cells were ready for implantation. A left fifth intercostal thoracotomy was performed, and 10 epicardial injections of the muscle preparation (between 10 and 20 million cells) were carried out. All sheep were euthanized 3 weeks after myocardial implantation. Immunohistochemistry was performed with monoclonal antibodies to a fast skeletal isoform of myosin heavy chain. Skeletal myosin heavy-chain expression was detected in all slides at 3 weeks after implantation in 8 of 8 animals, confirming engraftment of skeletal muscle cells. Massive areas of engraftment (from 2 to 9 mm in diameter) or discrete loci were noted within the myocardial wall. CONCLUSIONS: Our results indicate that noncultured skeletal muscle cells can successfully and massively engraft in ovine myocardium. Thus, avoiding the cell culture expansion phase is feasible and could become a promising option for cellular cardiomyoplasty.