We have previously developed bioengineered three-dimensional internal anal sphincter (IAS) rings from circular smooth muscle cells isolated from rabbit and human IAS. We provide proof of concept that bioengineered mouse IAS rings are neovascularized upon implantation into mice of the same strain and maintain concentric smooth muscle alignment, phenotype, and IAS functionality. Rings were bioengineered by using smooth muscle cells from the IAS of C57BL/6J mice. Bioengineered mouse IAS rings were implanted subcutaneously on the dorsum of C57BL/6J mice along with a microosmotic pump delivering fibroblast growth factor-2. The mice remained healthy during the period of implantation, showing no external signs of rejection. Mice were killed 28 days postsurgery and implanted IAS rings were harvested. IAS rings showed muscle attachment, neovascularization, healthy color, and no external signs of infection or inflammation. Assessment of force generation on harvested IAS rings showed the following: 1) spontaneous basal tone was generated in the absence of external stimulation; 2) basal tone was relaxed by vasoactive intestinal peptide, nitric oxide donor, and nifedipine; 3) acetylcholine and phorbol dibutyrate elicited rapid-rising, dose-dependent, sustained contractions repeatedly over 30 min without signs of muscle fatigue; and 4) magnitudes of potassium chloride-induced contractions were 100% of peak maximal agonist-induced contractions. Our preliminary results confirm the proof of concept that bioengineered rings are neovascularized upon implantation. Harvested rings maintain smooth muscle alignment and phenotype. Our physiological studies confirm that implanted rings maintain 1) overall IAS physiology and develop basal tone, 2) integrity of membrane ionic characteristics, and 3) integrity of membrane associated intracellular signaling transduction pathways for contraction and relaxation by responding to cholinergic, nitrergic, and VIP-ergic stimulation. IAS smooth muscle tissue could thus be bioengineered for the purpose of implantation to serve as a potential graft therapy for dysfunctional internal anal sphincter in fecal incontinence.
Pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, have generated much excitement about their prospects for use in cell transplantation therapies. This is largely attributable to their virtually unlimited growth potential, their ability to be precisely genetically altered in culture, and their utility for forming differentiated cell populations with potential clinical applications. Lysosomal storage diseases such as Sanfilippo syndrome (MPS-IIIA) represent ideal candidate diseases for the evaluation of cell therapies in the central nervous system (CNS). These diseases exhibit widespread pathology yet result from a single gene deficiency, in the case of Sanfilippo syndrome the lysosomal enzyme sulfamidase. The aim of this study was to investigate mouse embryonic stem (ES) cell-derived glial precursor cells as a vehicle for sulfamidase delivery in the MPS-IIIA mouse brain. In this study we have created a mouse ES cell line genetically modified to stably express and secrete high levels of human sulfamidase and a protocol for the in vitro derivation of large numbers glial precursors from ES cells. Differentiation of sulfamidase-expressing ES cells resulted in cell populations with sustained secretion of high levels of sulfamidase, comprised primarily of glial precursor cells with minor contaminants of other neural cell phenotypes but not residual pluripotent cells. CNS implantation studies demonstrated that ES cell-derived glial precursor cells formed using this differentiation method were able to engraft and survive for at least 12 weeks following implantation. The percentage of engraftment was quantified in different regions of the brain in 2-, 4-, and 8-week-old normal and MPS-IIIA mice. No teratomas were observed in any of the cell-transplanted animals. The results of this study support the further investigation of sulfamidase-expressing glial precursor cells as a vehicle for delivery of deficient enzyme into the CNS of MPS-IIIA mice.
