Raghavan Shreya, Bitar Khalil N
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27101, USA; Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem, NC 27101, USA.
Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27101, USA; Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston-Salem, NC 27101, USA.
Biomaterials. 2014 Aug;35(26):7429-40. doi: 10.1016/j.biomaterials.2014.05.037. Epub 2014 Jun 11.
Differentiation of enteric neural stem cells into several appropriate neural phenotypes is crucial while considering transplantation as a cellular therapy to treat enteric neuropathies. We describe the formation of tissue engineered innervated sheets, where intestinal smooth muscle and enteric neuronal progenitor cells are brought into close association in extracellular matrix (ECM) based microenvironments. Uniaxial alignment of constituent smooth muscle cells was achieved by substrate microtopography. The smooth muscle component of the tissue engineered sheets maintained a contractile phenotype irrespective of the ECM composition, and generated equivalent contractions in response to potassium chloride stimulation, similar to native intestinal tissue. We provided enteric neuronal progenitor cells with permissive ECM-based compositional and viscoelastic cues to generate excitatory and inhibitory neuronal subtypes. In the presence of the smooth muscle cells, the enteric neuronal progenitor cells differentiated to functionally innervate the smooth muscle. The differentiation of specific neuronal subtypes was influenced by the ECM microenvironment, namely combinations of collagen I, collagen IV, laminin and/or heparan sulfate. The physiology of differentiated neurons within tissue engineered sheets was evaluated. Sheets with composite collagen and laminin had the most similar patterns of Acetylcholine-induced contraction to native intestinal tissue, corresponding to an increased protein expression of choline acetyltransferase. An enriched nitrergic neuronal population, evidenced by an increased expression of neuronal nitric oxide synthase, was obtained in tissue engineered sheets that included collagen IV. These sheets had a significantly increased magnitude of electrical field stimulated relaxation, sensitive maximally to nitric oxide synthase inhibition. Tissue engineered sheets containing laminin and/or heparan sulfate had a balanced expression of contractile and relaxant motor neurons. Our studies demonstrated that neuronal subtype was modulated by varying ECM composition. This observation could be utilized to derive enriched populations of specific enteric neurons in vitro prior to transplantation.
在考虑将移植作为治疗肠道神经病变的细胞疗法时，肠神经干细胞分化为几种合适的神经表型至关重要。我们描述了组织工程化神经支配片的形成，其中肠道平滑肌和肠神经元祖细胞在基于细胞外基质（ECM）的微环境中紧密结合。通过底物微观形貌实现了组成平滑肌细胞的单轴排列。组织工程片的平滑肌成分无论ECM组成如何都保持收缩表型，并在氯化钾刺激下产生与天然肠道组织相似的等效收缩。我们为肠神经元祖细胞提供基于ECM的允许性组成和粘弹性线索，以产生兴奋性和抑制性神经元亚型。在平滑肌细胞存在的情况下，肠神经元祖细胞分化以功能性地支配平滑肌。特定神经元亚型的分化受ECM微环境的影响，即I型胶原、IV型胶原、层粘连蛋白和/或硫酸乙酰肝素的组合。评估了组织工程片内分化神经元的生理学。含有复合胶原和层粘连蛋白的片对乙酰胆碱诱导的收缩模式与天然肠道组织最相似，对应于胆碱乙酰转移酶蛋白表达增加。在包含IV型胶原的组织工程片中获得了丰富的含氮能神经元群体，这通过神经元型一氧化氮合酶表达增加得到证明。这些片电场刺激松弛的幅度显著增加，对一氧化氮合酶抑制最为敏感。含有层粘连蛋白和/或硫酸乙酰肝素的组织工程片具有收缩性和舒张性运动神经元的平衡表达。我们的研究表明，神经元亚型受不同ECM组成的调节。这一观察结果可用于在移植前体外获得富集的特定肠神经元群体。
