Mechanisms in Choroid Plexus Epithelial Development

Mechanisms in Choroid Plexus Epithelial Development

Funding Type: 
New Faculty II
Grant Number: 
RN2-00915
Award Value: 
$2,793,395
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Active
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

Our project goals are to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then apply this information to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first Aim is to determine whether a factor known as Fgf8 promotes CPe fate, the second Aim addresses whether the Lhx2 transcription factor inhibits CPe, and the third Aim is to generate human CPe cells in culture. Significant progress on these Aims has been made during this first year of the grant. Most importantly, multiple lines of evidence for CPe differentiation from both mouse and human ESCs have been obtained. In addition, the genetically-engineered mESC lines needed for the Lhx2 studies in Aim 2 have been successfully generated and validated. Our major goals for the next year are to further replicate, confirm, and optimize the generation of CPe cells in our mouse and human ESC cultures, and to perform the initial experiments that should determine whether manipulating Fgf8 and Lhx2 in the ESC cultures will enhance CPe generation in culture.

Year 2

Our goal is to define the factors involved in choroid plexus epithelial (CPe) cell development in mice, then to apply this knowledge to generate CPe cells from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims examine Fgf8 and Lhx2 as promoter and inhibitor, respectively, of CPe fate, and the third Aim is to generate human CPe cells in culture. Unexpectedly, we obtained significant evidence for CPe differentiation from both mouse and human ESCs during year 1 of the award. Our aims for year 2 were therefore modified to accelerate the translation of our findings towards a CPe-based regenerative medicine. This year, we developed a second cell culture system for deriving mouse CPe cells, and established a functional assay for CPe cells in culture, which we used to confirm the function of our derived mouse CPe cells. To sort and purify CPe cells for clinical applications, we began characterizing CPe cell complexity, size, and mitochondrial content by flow cytometry, obtained a mouse line with fluorescent CPe cells, and identified three antibodies that may be useful for sorting human CPe cells. A stereotaxic injection system was built, and institutional approvals were obtained, to establish methods for replacing or transplanting CPe cells in the mouse brain.

Year 3

The goal of this project is to define the factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. The first two Aims used mice to examine a potential promoter and inhibitor, respectively, of CPEC fate, and the third Aim is to generate human CPECs in culture. Unexpectedly early success in CPEC derivation from human ESCs has allowed us to accelerate Aim 3 and the pursuit of translational goals this year. We further optimized our existing human CPEC derivation method and developed a second method (a combined suspension-adherent system) that may prove to be much more efficient. Several new GMP-compliant human ESC lines were approved and obtained. To facilitate the translational efforts, we made many new mouse ESC lines that were designed to fluoresce when CPECs are produced, and this was confirmed using the first of these lines. A crude CPEC purification strategy was also developed, and using this strategy, transplantation of partially-purified CPECs into mice was established in the lab this year. Remarkably, we found that transplanted mESC-derived CPECs, on their own, can integrate into endogenous choroid plexus with relatively high efficiency. This opens up several new and exciting therapeutic possibilities. To further enhance choroid plexus engraftment, a mouse CPEC ablation approach is currently being tested. A collaboration was initiated to profile all of the genes expressed by the purified mouse ESC-derived CPECs, and to compare this profile to those expressed by the choroid plexus in developing mice and humans. Industry partnerships and non-provisional patenting were also pursued to enhance the prospects for human CPEC applications in drug screening and treating patients with a wide range of neurodegenerative and other nervous system disorders.

Year 4

The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We tested two new culture systems, with promising results from a more controllable and scalable monolayer culture system that will facilitate the improvement of dCPEC generation efficiency. New transcriptome profiling studies allowed us to better define highly-expressed genes for cell surface proteins, which will be targeted to purify dCPECs for downstream applications. New double-labelling and whole mount preparations of mouse choroid plexus have been devised to facilitate ongoing efforts to improve dCPEC engraftment of host choroid plexus after injection, and a new functional assay for dCPEC barrier formation and regulation has been established to complement an already-existing functional secretion assay in the lab. Efforts are also now underway to generate fluorescent and luminescent CPEC reporter hESC lines that should greatly facilitate dCPEC process development (derivation and purification). During this past year, new industry partners were recruited, an initial paper describing the dCPEC technology was published, and an initial patent application on the dCPEC technology was filed.

Year 5

The goal of this project is to define factors involved in choroid plexus epithelial cell (CPEC) development in mice, then to apply this knowledge to generate CPECs from mouse and human embryonic stem cells (ESCs) for clinical applications. Unexpected early success in generating ESC-derived CPECs (dCPECs) allowed us to accelerate and focus on the more translational goals of the project this year. We further developed two culture systems - a more controllable monolayer system and more scalable rotational aggregate system - that will facilitate the dCPEC work. After several disappointments, improvements in dCPEC differentiation efficiency were obtained with two pharmacologic agents. With help from transcriptome profiling studies, we identified cell surface proteins that could be utilized for dCPEC enrichment, with initial promising results for one candidate surface antigen. A robust whole mount choroid plexus culture system was newly developed to facilitate efforts to improve dCPEC engraftment of host choroid plexus, and methods surrounding the stereotactic injection of dCPECs have been improved. After some difficulties, human TTR BAC constructs that express fluorescent and luminescent reporters were created and validated; these will be used to generate new CPEC reporter mouse lines for endpoint and longitudinal studies, and for in vivo drug testing of compounds that enhance TTR production and CPEC secretion. The initial patent application on the dCPEC technology was reviewed by the US PTO, and a revision was submitted.

© 2013 California Institute for Regenerative Medicine