Cellular expression of Noc2, a Rab effector protein, in endocrine and exocrine tissues in the mouse.

Noc2 is a Rab effector which participates in regulated exocytosis. It is expressed abundantly in endocrine cells but at low levels in exocrine tissues. Noc2-deficient mice, however, exhibit marked accumulation of secretory granules in exocrine cells rather than endocrine cells. In the present study, we investigated localization of Noc2 immunohistochemically in various endocrine and exocrine tissues in normal mice. Western blotting detected a Noc2-immunoreactive band of 38 kDa in isolated pancreatic islets, the adrenal gland, pituitary gland, and thyroid gland. Immunostaining for Noc2 labeled endocrine cells in the adrenal medulla and adenohypophysis, pancreatic islet cells, thyroid parafollicular cells, and gut endocrine cells, supporting the notion that Noc2 is a Rab effector protein shared by amine/peptide-secreting endocrine cells. Besides endocrine tissues, granular ducts in salivary glands contained Noc2. Although immunostaining failed to detect Noc2 in acinar cells of all exocrine glands examined, reverse transcriptase-polymerase chain reaction analysis detected the mRNA expression in exocrine pancreas. Ultrastructurally, Noc2 immunoreactivity was associated with the limiting membrane of granules in both pancreatic endocrine and salivary duct exocrine cells. The cellular and subcellular localizations of Noc2 should yield key information on its functional significance as well as account for the phenotype in Noc2-deficient mice

Smooth Muscle-Like Tissue Constructs with Circumferentially Oriented Cells Formed by the Cell Fiber Technology

<div><p>The proper functioning of many organs and tissues containing smooth muscles greatly depends on the intricate organization of the smooth muscle cells oriented in appropriate directions. Consequently controlling the cellular orientation in three-dimensional (3D) cellular constructs is an important issue in engineering tissues of smooth muscles. However, the ability to precisely control the cellular orientation at the microscale cannot be achieved by various commonly used 3D tissue engineering building blocks such as spheroids. This paper presents the formation of coiled spring-shaped 3D cellular constructs containing circumferentially oriented smooth muscle-like cells differentiated from dedifferentiated fat (DFAT) cells. By using the cell fiber technology, DFAT cells suspended in a mixture of extracellular proteins possessing an optimized stiffness were encapsulated in the core region of alginate shell microfibers and uniformly aligned to the longitudinal direction. Upon differentiation induction to the smooth muscle lineage, DFAT cell fibers self-assembled to coiled spring structures where the cells became circumferentially oriented. By changing the initial core-shell microfiber diameter, we demonstrated that the spring pitch and diameter could be controlled. 21 days after differentiation induction, the cell fibers contained high percentages of ASMA-positive and calponin-positive cells. Our technology to create these smooth muscle-like spring constructs enabled precise control of cellular alignment and orientation in 3D. These constructs can further serve as tissue engineering building blocks for larger organs and cellular implants used in clinical treatments.</p></div

Formation of DFAT cell fiber.

<p>(a) Schematic drawing of the microfluidic double co-axial device used to encapsulate cells into the core of alginate fibers with the default flow rates. The inner core stream consist of DFAT cells suspended in the appropriate ECM proteins. The outer shell stream is sodium alginate solution that solidifies when it comes in contact with the sheath stream of calcium chloride solution. (b) Cell fiber formation overnight. A day 0 image captured immediately following fiber fabrication with single DFAT cells dispersed in ECM proteins encapsulated in the center core of the alginate fiber, and a day 1 image showing a section of the formed DFAT cell fiber inside alginate shell. (c) Images comparing DFAT cell alignment when cultured in conventional 2D dish and as 3D fiber (day 2) structure. DFAT cells are randomly oriented when attached on 2D dishes, whereas DFAT cells are highly aligned along the longitudinal fiber direction when cultured as 3D fiber structure.</p

Overall concept of the study.

<p>DFAT cells derived from adipocytes are encapsulated into the center core of alginate fibers and allowed to form into cell fiber. DFAT cell fiber is subsequently induced for differentiation into smooth muscle cell lineage in induction media containing TGF-β. As cells’ inherent cell traction force increases during this process, linear cell fiber self-assembles to coiled spring structure. Such smooth muscle-like cell springs may serve as tissue engineering building blocks.</p

Core ECM protein optimization.

<p>(a) Bar graph of the average Young’s modulus of various types of ECM protein, organized in increasing stiffness. n = 50, and data are expressed as the mean ± standard deviation. (b) PCol and ACol-L failed to support fiber formation as shown in the day 1 images where DFAT cells mainly aggregated into spheroids as opposed to cell fibers. (c) ACol-H is stiff enough to support initial formation of DFAT cells into fiber structures, but failed to maintain the fiber structures as almost half of the cells already aggregated into spheroids by day 10. All concentrations of fibrin tested successfully supported initial DFAT cell fiber formation (day 1), but by day 33, almost all cells in Fibrin-L (d) and about half of the cells in Fibrin-M (e) aggregated to spheroids, and only Fibrin-H (f) still maintained the fiber structures. (g) Mixture of collagen and fibrin, ACol-Fib, is also able to support initial DFAT cell fiber formation as well as maintain the fiber structures for at least 33 days. Scale bar = 1 mm.</p