Assessment of Esophageal Reconstruction via Bioreactor Cultivation of a Synthetic Scaffold in a Canine Model

Objectives@#. Using tissue-engineered materials for esophageal reconstruction is a technically challenging task in animals that requires bioreactor training to enhance cellular reactivity. There have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in initial epithelialization in the special environment of peristalsis. The purpose of this study was to evaluate the potential of an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a double-layered polymeric scaffold and a custom-designed mesenchymal stem cell-based bioreactor system in a canine model. @*Methods@#. We fabricated a novel double-layered scaffold as a tissue-engineered esophagus using an electrospinning technique. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. After 3 days of cultivation using the bioreactor system, tissue-engineered artificial esophagus was transplanted into a partial esophageal defect (5×3 cm-long resection) in a canine model. @*Results@#. Scanning electron microscopy (SEM) showed that the electrospun fibers in a tubular scaffold were randomly and circumferentially located toward the inner and outer surfaces. Complete recovery of the esophageal mucosa was confirmed by endoscopic analysis and SEM. Esophagogastroduodenoscopy and computed tomography also showed that there were no signs of leakage or stricture and that there was a normal lumen with complete epithelialization. Significant regeneration of the mucosal layer was observed by keratin-5 immunostaining. Alpha-smooth muscle actin immunostaining showed significantly greater esophageal muscle regeneration at 12 months than at 6 months. @*Conclusion@#. Custom-designed bioreactor cultured electrospun polyurethane scaffolds can be a promising approach for esophageal tissue engineering.

Stem Cells Seeded on Multilayered Scaffolds Implanted into an Injured Bladder Rat Model Improves Bladder Function

BACKGROUND: To investigate whether human adipose-derived stem cells (hADSCs) seeded on multilayered poly (L-lactide-co-ε-caprolactone) (PLCL) sheets improve bladder function in a rat model of detrusor smooth muscle-removed bladder. METHODS: Male rats were randomly divided into 4 groups: Normal, injury (detrusor smooth muscle-removed bladder), PLCL (detrusor smooth muscle-removed bladder implanted with PLCL sheets), and PLCL + ADSC (detrusor smooth muscle-removed bladder implanted with PLCL sheets seeded with hADSCs). Four weeks after the treatment, physiological, histological, immunohistochemical, and immunoblot analyses were performed. RESULTS: hADSCs were compatible with PLCL sheets. Further, the physiological study of PLCL + ADSC group showed significant improvement in compliance and contractility suggesting the functional improvement of the bladder. Histological, immunohistochemical and immunoblot analyses revealed the uniform distribution of hADSCs in between PLCL sheets as well as differentiation of hADSCs into smooth muscle cells (SMC) which is illustrated by the expression of SMC markers. CONCLUSION: hADSCs seeded on the multilayered PLCL sheets has the potential to differentiate into SMC, thus facilitating the recovery of compliance and contractility of the injured bladder.

Directing Human Embryonic Stem Cells towards Functional Endothelial Cells Easily and without Purification

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Comparison of Human Muscle-Derived Stem Cells and Human Adipose-Derived Stem Cells in Neurogenic Trans-Differentiation

PURPOSE: Erectile dysfunction (ED) remains a major complication from cavernous nerve injury during radical prostatectomy. Recently, stem cell treatment for ED has been widely reported. This study was conducted to investigate the availability, differentiation into functional cells, and potential of human muscle-derived stem cells (hMDSCs) and human adipose-derived stem cells (hADSCs) for ED treatment. MATERIALS AND METHODS: We compared the neural differentiation of hMDSCs and hADSCs. Human muscle and adipose tissues were digested with collagenase, followed by filtering and centrifugation. For neural induction, isolated hMDSCs and hADSCs were incubated in neurobasal media containing forskolin, laminin, basic-fibroblast growth factor, and epidermal growth factor for 5 days. Following neural induction, hMDSCs and hADSCs were differentiated into neural cells, including neurons and glia, in vitro. RESULTS: In neural differentiated hMDSCs (d-hMDSCs) and differentiated hADSCs (d-hADSCs), neural stem cell marker (nestin) showed a significant decrease by immunocytochemistry, and neuronal marker (beta-tubulin III) and glial marker (GFAP) showed a significant increase, compared with primary hMDSCs and hADSCs. Real-time chain reaction analysis and Western blotting demonstrated significantly elevated levels of mRNA and protein of beta-tubulin III and GFAP in d-hADSCs compared with d-hMDSCs. CONCLUSIONS: We demonstrated that hMDSCs and hADSCs can be induced to undergo phenotypic and molecular changes consistent with neurons. The neural differentiation capacity of hADSCs was better than that of hMDSCs.