Collaborative work in between the chemical engineering and cell biology departments will identify conditions to build an in vitro model to study normal and pathological neural development
- Tissue Engineering and Regenerative Medicine
- Stem cell and development
BTBernard V. ThisseSchool of Medicine
KLKyle J. LampeSchool of Engineering and Applied Science
RLRachel A LetteriSchool of Engineering and Applied Science
Creating in vitro models of neural development amenable to experimentalmanipulation and drug screening is critically important for delineating themolecular basis of, and developing clinical therapies for, neurological disorders thataffect more than one billion people worldwide. Toward this goal, three-dimensional neuralorganoids that display some structuraland functional features of neural tissues have been developed. However, whilethese organoids exhibit microanatomy similar to the authentic brain, they lackproper patterning and interactions with surrounding non-neural tissues. Thus, theneural organoids developed, to date, are unable to receive input from sensoryorgans and disseminate signals to the rest of the body. Therefore, they cannotmimic a fully functional nervous system, greatly limiting their use. We seek torectify this deficiency by building an in vitro embryo-mimetic system that contains a fully patterned neuraltube, capable of receiving signals, andrelaying them systemically. Our innovative strategy is to initiate embryonicdevelopment in embryoid bodies (EBs), generated from mouse embryonic stem cells(ESCs), instructed with spatially patterned morphogen gradients. Ourpreliminary studies show that these instructed EBs develop into “embryoids”that gastrulate and form the three patterned, bilaterally symmetrical germlayers. Theembryoids also contain a neural plate comprised of columnar neuroepithelialcells that progressively folds into a neural tube. While hindbrain and spinalcord are present, the embryoids lackthe anterior most part of the brain, forebrain and midbrain. Wehypothesize that this absence of anterior brain domains results from the excessof instructive signaling secreted by the organizing morphogen activity centerwe apply, and that correcting this problemwill optimize our in vitro model forthe study of normal and pathological neural development.
Our goal is to identify experimental conditions where the activitygradient secreted by the organizing center will be negatively regulated throughan opposing gradient of specific antagonist(s). To achieve this goal, wepropose to engineer microparticle centers from which antagonist(s) will diffuse.This will allow modifying the shape, timing, and extent of the activitygradient secreted by the organizing center. Specifically, we will deliverprotein antagonist(s) with control of temporal release by using poly (lactic-co-glycolic acid) (PLGA) microparticles.PLGA microparticles are commonly synthesized via a water/oil/water emulsion method in the Lampe Group and usedto deliver proteins on the scale of hours to weeks. Release rate is controlledby adjusting the molecular weight and lactide: glycolide ratio of the PLGApolymer. These microparticles will then be modified with the expertise of Prof.Letteri to vary surface charge and composition in order to facilitateattachment to ESCs and integration into EBs. Target surface coatings willinclude hydrophilic small molecules and polymers that impart cationic, anionic,or neutral surface charge and promote cell adhesion. Furthermore, a variety ofmodifications will be explored in an effort to simplify the particlefabrication process, while providing tunable particles for delivery of antagonists.
Altogether, we predict that the outcome of this approach will be theformation of embryoids displaying a complete neural tube, including the telencephalondown to the posterior part of the spinal cord. We will therefore provide thefirst complete model of mammalian neural development in vitro that will also laythe groundwork for the creation in the future of an autologous human nervoussystem model, using human induced pluripotent stem cells. The methodologyemployed will be novel and scalable, readily transferable to other researchersand corporations.
Create the first complete model of mammalianneural development in vitro
1) We will modify PLGA particles, both covalently and non-covalently, torender the surface cationic, anionic, or neutral to elucidate the impact ofsurface charge on cell attachment.
2) We will vary the amount of residual PVA surfactant during thefabrication process as a simpler method to vary surface charge.
3) We will utilize the reactive functional groups on PVA-stabilized PLGAmicroparticles to non-covalently modify the surfaces in an effort to diversifythe surface composition and particle-cell interactions.
4) We will investigate the impact of cell-adhesive peptide ligands onmicroparticle attachment.
5) We will probe embryoids at various developmental stages (D7-D11) forthe expression and the relative position of forebrain, midbrain and hindbrainmarkers, analyzed by multicolor fluorescent or chromogenic in situ hybridization.
45K will be used for salary support (Post-doc Dr Ricardo Borges, GRANick Murphy, GRA to be named Oct. 12). The rest of the funds (15K) will be usedto acquire the necessary materials, cell culture reagents, and the use ofparticle characterization facilities (SEM imaging) for this project.