Roger E. De Filippo

Urethral Replacement Using Cell Seeded Tubularized Collagen Matrices

PURPOSE Acellular collagen matrices derived from bladder submucosa have been used successfully as an off-the-shelf biomaterial for urethral replacement, experimentally and clinically in an onlay fashion. We investigated whether collagen matrices, either alone or with autologous cells, could be used for tubularized urethral replacement. MATERIALS AND METHODS Acellular collagen matrices were processed and tubularized. Ten rabbits underwent an open bladder biopsy with subsequent cell expansion. Autologous bladder cells were grown and seeded onto the pre-configured tubular matrices. A 1 cm. long urethral segment was excised in 24 male rabbits. Urethroplasty was performed with the tubularized collagen matrices seeded with cells in 12 animals and without cells in 12. Serial urethrography was performed preoperatively and at 1, 2, 3 and 6 months postoperatively. Retrieved urethras were analyzed grossly, histologically, immunocytochemically and with Western blots. Contractility and the presence of neurotransmitter receptors were confirmed with organ bath studies. RESULTS Serial urethrography confirmed the maintenance of a wide urethral caliber without any signs of strictures in animals implanted with the cell seeded matrices. The urethral segments replaced with the collagen scaffolds without cells demonstrated strictures and graft collapse at all time points. The implanted cell seeded matrices had a normal urethral architecture by 1 month, consisting of a transitional cell layer surrounded by muscle cell fiber bundles with increasing cellular organization with time. Epithelial and smooth muscle phenotypes were confirmed immunocytochemically and with Western blot analyses using pancytokeratins AE1/AE3 and smooth muscle specific alpha-actin antibodies. Formation of a transitional cell layer was confirmed in the matrices implanted without cells but only scant unorganized muscle fiber bundles were present, mostly at the anastomotic sites. Organ bath studies demonstrated the capacity for contractility along with cholinergic and adrenergic specific receptors in the tissue engineered scaffolds compared to controls. CONCLUSIONS These results show that collagen matrices seeded with cells form normal urethral tissue can be used for tubularized replacement, whereas tubularized collagen matrices alone without cells lead to poor tissue formation and strictures. The collagen matrices seeded with cells may offer a useful alternative in the future for patients requiring a tubularized urethral segment replacement.

Human Amniotic Fluid Stem Cells Can Integrate and Differentiate into Epithelial Lung Lineages

A new source of stem cells has recently been isolated from amniotic fluid; these amniotic fluid stem cells have significant potential for regenerative medicine. These cells are multipotent, showing the ability to differentiate into cell types from each embryonic germ layer. We investigated the ability of human amniotic fluid stem cells (hAFSC) to integrate into murine lung and to differentiate into pulmonary lineages after injury. Using microinjection into cultured mouse embryonic lungs, hAFSC can integrate into the epithelium and express the early human differentiation marker thyroid transcription factor 1 (TTF1). In adult nude mice, following hyperoxia injury, tail vein‐injected hAFSC localized in the distal lung and expressed both TTF1 and the type II pneumocyte marker surfactant protein C. Specific damage of Clara cells through naphthalene injury produced integration and differentiation of hAFSC at the bronchioalveolar and bronchial positions with expression of the specific Clara cell 10‐kDa protein. These results illustrate the plasticity of hAFSC to respond in different ways to different types of lung damage by expressing specific alveolar versus bronchiolar epithelial cell lineage markers, depending on the type of injury to recipient lung.

Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis

Acute Tubular Necrosis (ATN) causes severe damage to the kidney epithelial tubular cells and is often associated with severe renal dysfunction. Stem-cell based therapies may provide alternative approaches to treating of ATN. We have previously shown that clonal c-kitpos stem cells, derived from human amniotic fluid (hAFSC) can be induced to a renal fate in an ex-vivo system. Herein, we show for the first time the successful therapeutic application of hAFSC in a mouse model with glycerol-induced rhabdomyolysis and ATN. When injected into the damaged kidney, luciferase-labeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN. We therefore speculate that AFSC could represent a novel source of stem cells that may function to modulate the kidney immune milieu in renal failure caused by ATN.

Engineering of vaginal tissue in vivo.

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.

Human Amniotic Fluid as a Potential New Source of Organ Specific Precursor Cells for Future Regenerative Medicine Applications

PURPOSE Human amniotic fluid contains multiple cell types, including pluripotent and committed progenitor cells, and fully differentiated cells. We characterized various cell populations in amniotic fluid. MATERIALS AND METHODS Optimum culture techniques for multiple cell line passages with minimal morphological change were established. Cell line analysis and characterization were done with reverse transcriptase and real-time polymerase chain reaction. Immunoseparation was done to distinguish native progenitor cell lines and their various subpopulations. RESULTS Endodermal and mesodermal marker expression was greatest in samples of early gestational age while ectodermal markers showed a constant rate across all samples. Pluripotent and mesenchymal cells were always present but hematopoietic cell markers were expressed only in older samples. Specific markers for lung, kidney, liver and heart progenitor cells were increasingly expressed after 18 weeks of gestation. We specifically focused on a CD24+OB-cadherin+ population that could identify uninduced metanephric mesenchyma-like cells, which in vivo are nephron precursors. The CD24+OB-cadherin+ cell line was isolated and subjected to further immunoseparation to select 5 distinct amniotic fluid kidney progenitor cell subpopulations based on E-cadherin, podocalyxin, nephrin, TRKA and PDGFRA expression, respectively. CONCLUSIONS These subpopulations may represent different precursor cell lineages committed to specific renal cell fates. Committed progenitor cells in amniotic fluid may provide an important and novel resource of useful cells for regenerative medicine purposes.

Autologous cell seeded biodegradable scaffold for augmentation cystoplasty: phase II study in children and adolescents with spina bifida.

PURPOSE Augmentation cystoplasty using gastrointestinal segments in children/adolescents with medically refractory neurogenic bladder is associated with significant complications. We evaluated an autologous cell seeded biodegradable scaffold (Tengion®) for bladder augmentation as an alternative to traditional enterocystoplasty in this population. MATERIALS AND METHODS A phase II prospective study was performed in children with neurogenic bladder due to spina bifida requiring enterocystoplasty for detrusor pressure 40 cm H2O or greater despite maximum antimuscarinic medication. Following open bladder biopsy, urothelial and smooth muscle cells were grown ex vivo and seeded onto a biodegradable scaffold to form a regenerative augment as the foundation for bladder tissue regeneration. Bladder neck sling was the only concomitant surgical procedure permitted. Bladders were cycled postoperatively to promote regeneration. Primary and secondary outcomes at 12 months included change in bladder compliance, bladder capacity and safety. Long-term assessment was done with similar outcomes at 36 months. RESULTS Compliance improved in 4 patients at 12 months and in 5 patients at 36 months, although the difference was not clinically or statistically significant. There was no clinical or statistical improvement in bladder capacity at 12 or 36 months in any patient. Adverse events occurred in all patients, and most were easily treated. Two patients had low cell growth following bladder biopsy, of whom 1 withdrew from the study and 1 underwent a second biopsy. Serious adverse events of bowel obstruction and/or bladder rupture occurred in 4 patients. CONCLUSIONS Our autologous cell seeded biodegradable scaffold did not improve bladder compliance or capacity, and our serious adverse events surpassed an acceptable safety standard.

Characterization of Human Amniotic Fluid Stem Cells and Their Pluripotential Capability

Over the past decade, there has been ever-increasing emphasis placed on stem cells and their potential role in regenerative medicine for reconstruction of bio-artificial tissues and organs. Scientists have looked at various sources for pluripotential cells ranging from embryonic stem cells to adult stem cells. Amniocentesis is a well-established technique for the collection of cells derived from the human embryo. In this chapter, we are going to describe how to isolate, maintain in culture, and characterize the pluripotential capabilities of stem cells derived from amniocentesis in an in vitro and in vivo system. Cell samples are obtained from human pregnancies, and the progenitor cells are isolated from male fetuses with a normal karyotype in order to confirm the absence of maternal admixed cells. Progenitor cells express embryonic-specific cell markers, they show a high self-renewal capacity with 350 population doublings, and normal ploidy is confirmed by cell-cycle analyses. They maintain their undifferentiated state, pluripotential ability, clonogenicity, and telomere length over the population doublings. The progenitor cells are inducible to different cell lineages (osteogenic, adipogenic, skeletal muscle, endothelial, neuronal, and hepatic cells) under specific growth conditions. The ability to induce cell-type-specific differentiation is confirmed by phenotypic changes, immunocytochemistry, gene expression, and functional analyses. In addition, we will describe an application of these cells in an ex vivo and in vivo system for potential in organ (renal) regeneration. The progenitor cells described in this chapter have a high potential for expansion, and may be a good source for research and therapeutic applications where large numbers of cells are needed. Progenitor cells isolated during gestation may be beneficial for fetuses diagnosed with malformations and could be cryopreserved for future self-use.

Injection of Amniotic Fluid Stem Cells Delays Progression of Renal Fibrosis

Injection of amniotic fluid stem cells ameliorates the acute phase of acute tubular necrosis in animals by promoting proliferation of injured tubular cells and decreasing apoptosis, but whether these stem cells could be of benefit in CKD is unknown. Here, we used a mouse model of Alport syndrome, Col4a5(-/-) mice, to determine whether amniotic fluid stem cells could modify the course of progressive renal fibrosis. Intracardiac administration of amniotic fluid stem cells before the onset of proteinuria delayed interstitial fibrosis and progression of glomerular sclerosis, prolonged animal survival, and ameliorated the decline in kidney function. Treated animals exhibited decreased recruitment and activation of M1-type macrophages and a higher proportion of M2-type macrophages, which promote tissue remodeling. Amniotic fluid stem cells did not differentiate into podocyte-like cells and did not stimulate production of the collagen IVa5 needed for normal formation and function of the glomerular basement membrane. Instead, the mechanism of renal protection was probably the paracrine/endocrine modulation of both profibrotic cytokine expression and recruitment of macrophages to the interstitial space. Furthermore, injected mice retained a normal number of podocytes and had better integrity of the glomerular basement membrane compared with untreated Col4a5(-/-) mice. Inhibition of the renin-angiotensin system by amniotic fluid stem cells may contribute to these beneficial effects. In conclusion, treatment with amniotic fluid stem cells may be beneficial in kidney diseases characterized by progressive renal fibrosis.

Human capital gains associated with robotic assisted laparoscopic pyeloplasty in children compared to open pyeloplasty.

PURPOSE Robotic assisted laparoscopic pyeloplasty is an emerging, minimally invasive alternative to open pyeloplasty in children for ureteropelvic junction obstruction. The procedure is associated with smaller incisions and shorter hospital stays. To our knowledge previous outcome analyses have not included human capital calculations, especially regarding loss of parental workdays. We compared perioperative factors in patients who underwent robotic assisted laparoscopic and open pyeloplasty at a single institution, especially in regard to human capital changes, in an institutional cost analysis. MATERIALS AND METHODS A total of 44 patients 2 years old or older from a single institution underwent robotic assisted (37) or open (7) pyeloplasty from 2008 to 2010. We retrospectively reviewed the charts to collect demographic and perioperative data. The human capital approach was used to calculate parental productivity losses. RESULTS Patients who underwent robotic assisted laparoscopic pyeloplasty had a significantly shorter average hospital length of stay (1.6 vs 2.8 days, p <0.05). This correlated with an average savings of lost parental wages of

Stem cell and regenerative science applications in the development of bioengineering of renal tissue.

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