Recombinant laminin fragments endowed with collagen-binding activity: A tool for conferring laminin-like cell-adhesive activity to collagen matrices.

Laminins are major components of basement membranes that sustain a wide variety of stem cells. Among 15 laminin isoforms, laminin-511 and its E8 fragment (LM511E8) have been shown to strongly promote the adhesion and proliferation of human pluripotent stem cells. The aim of this study was to endow the cell-adhesive activity of laminin-511 on collagen matrices, thereby fabricating collagen-based culture scaffolds for stem cells with defined composition. To achieve this goal, we utilized the collagen-binding domain (CBD) of fibronectin to immobilize LM511E8 on collagen matrices. CBD was attached to the N-termini of individual laminin chains (α5E8, ß1E8, γ1E8), producing LM511E8s having one, two, or three CBDs. While LM511E8 did not bind to collagen, CBD-attached LM511E8s (CBD-LM511E8s) exhibited significant collagen-binding activity, dependent on the number of attached CBDs. Human iPS cells were cultured on collagen-coated plates preloaded with CBD-LM511E8s. Although iPS cells did not attach or grow on collagen, they robustly proliferated on CBD-LM511E8-loaded collagen matrices, similar to the case with LM511E8-coated plates. Importantly, iPS cells proliferated and yielded round-shaped colonies even on collagen gels preloaded with CBD-LM511E8s. These results demonstrate that CBD-attached laminin E8 fragments are promising tools for fabrication of collagen-based matrices having the cell-adhesive activity of laminins.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and schwann cell-like cells in a slow-flow microfluidic device.

BACKGROUND: To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived "neurons" could potentially substitute for lost or damaged SGN. mESC-derived "Schwann cells" produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a "cell-coated" implant. RESULTS: Neuron- and Schwann cell-like cells were produced from a common population of mESCs in an ultra-slow-flow microfluidic device. As the populations interacted, "neurons" grew over the "Schwann cell" lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing "Schwann cells" were used to coat a CI: Mouse SGN and MIF-induced "neurons" grew directionally to the CI and to a wild-type but not MIF-knockout organ of Corti explant. CONCLUSIONS: Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7-27, 2017. © 2016 Wiley Periodicals, Inc.

Effects of mouse utricle stromal tissues on hair cell induction from induced pluripotent stem cells.

BACKGROUND: Hair cells are important for maintaining our sense of hearing and balance. However, they are difficult to regenerate in mammals once they are lost. Clarification of the molecular mechanisms underlying inner ear disorders is also impeded by the anatomical limitation of experimental access to the human inner ear. Therefore, the generation of hair cells, possibly from induced pluripotent stem (iPS) cells, is important for regenerative therapy and studies of inner ear diseases. RESULTS: We generated hair cells from mouse iPS cells using an established stepwise induction protocol. First, iPS cells were differentiated into the ectodermal lineage by floating culture. Next, they were treated with basic fibroblast growth factor to induce otic progenitor cells. Finally, the cells were co-cultured with three kinds of mouse utricle tissues: stromal tissue, stromal tissue + sensory epithelium, and the extracellular matrix of stromal tissue. Hair cell-like cells were successfully generated from iPS cells using mouse utricle stromal tissues. However, no hair cell-like cells with hair bundle-like structures were formed using other tissues. CONCLUSIONS: Hair cell-like cells were induced from mouse iPS cells using mouse utricle stromal tissues. Certain soluble factors from mouse utricle stromal cells might be important for induction of hair cells from iPS cells.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear.

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.

A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation.

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/-), or null (-/-) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant.