Predifferentiation of human embryonic stem cells for CNS cortical applications

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Central nervous system (CNS) disorders such as those that affect the brain and eye are particularly debilitating because repair is hampered by the fact that neurons don’t divide to replace damaged areas and adult neural stem cells that can divide and potentially replace neurons don’t do so in the adult (for reasons we don’t understand). Therefore, it is intriguing to think that disorders such as stroke and macular degeneration of the eye may benefit from stem cell therapy. Treatment with stem cells derived from human embryonic stem cells (hESCs) requires pre-formation of a particular cell type, since transplantation of hESCs themselves leads to tumor formation. However, current methods for making CNS neurons are inadequate since the neurons are usually immature and unable to connect with the other neurons in the damaged area. Interestingly, treatment of animal models of CNS disorders with stem cells has shown some limited benefits without generation of mature neurons, possibly due to factors that the cells produce. However, transplantation of brain- or eye-specific cells derived from hESCs may lead to greater functional recovery (e.g. treatment of the damaged brain with a brain-specific neuron able to make functional connections with the host tissue). Certain regions of the brain (cortical) and eye (retinal) arise from the same area during development and share expression of a variety of markers, including regulatory proteins called transcription factors. Thus, it should be possible to make brain and eye cells from hESCs using similar conditions. Our goal is to use transcription factors to preferentially form brain and eye cells from hESCs. In one set of experiments, we will analyze transcription factors made by the hESCs in response to exposure to different conditions. In our second set of experiments, we will put transcription factors into hESCs during differentiation to test whether a particular transcription factor can direct formation of brain or eye neurons. This approach was recently shown to improve generation of the particular type of neurons damaged in Parkinson’s disease from mouse ESCs. We hope to use the cells we make for transplantation and also for drug testing, since there is currently no large-scale source of human versions of brain and eye neurons for these purposes.
Statement of Benefit to California: 
The goal of this project is to make brain- and eye-specific cells from hESCs that can be used for transplantation for disorders that affect specific brain regions (such as stroke) or the eye (retinal disorders such as macular degeneration). We also expect that the cells derived in this project could be used as a source of human brain or eye neurons for drug testing. Currently, there is no large-scale supply of such neurons. As such, if the goals of this project are realized, the benefits to the State of California and its citizens would include a greater range of therapeutic options for central nervous system disorders as well as an important source of cells for pharmaceutical and biotechnology companies.
Progress Report: 
  • CIRM Grant – Public Abstract:
  • Non-invasive imaging techniques for an in vivo tracking of transplanted stem cells offer real-time insight into the underlying biological processes of new stem cell based therapies, with the aim to depict stem cell migration, homing and engraftment at organ, tissue and cellular levels. We showed in previous experiments, that stem cells can be labeled effectively with contrast agents and that the labeled cells can be tracked non-invasively and repetitively with magnetic resonance imaging (MRI) and Optical imaging (OI). The purpose of this study was to apply and optimize these labeling techniques for a sensitive depiction of human embryonic stem cells (hESC) with OI and MRI.
  • Experimental Design: hESC were labeled with various contrast agents for MRI and OI, using a variety of labeling techniques, different contrast agent concentrations and different labeling intervals (1h – 24h). The cellular contrast agent uptake was proven by mass spectrometry (quantifies the iron oxides) and fluorescence microscopy (detects fluorescent dyes). The labeled hESC underwent imaging studies and extensive studies of their viability and ability to differentiate into specialized cell types.
  • Imaging studies: Decreasing numbers of 1 x 10^5 - 1 x 10^2 contrast agent-labeled hESC and non-labeled controls were evaluated with OI and MRI in order to determine the best contrast agent and labeling technique as well as the minimal detectable cell number with either imaging technique. In addition, samples of hESC were investigated with OI and MRI at 1 min, 2 min, 5 min, 1h, 2h, 6h, 12h, 24h and 48 h in order to investigate the stability of the label over time. Viability and differentiation assays of the hESC were performed before and after the labeling procedure in order to prove an unimpaired viability and function of the labeled cells.
  • Results: The FDA-approved contrast agents ferumoxides and indocyanine green (ICG) provided best results for MR and optical imaging (OI) applications. The cellular load with these labels was optimized towards the minimal concentration that allowed for detection with MR and OI, but did not alter cell viability or differentiation capacity. The ferumoxides and ICG-labeled hESCs as well as stem cell derived cardiomyocytes and chondrocytes provided significantly increased MR and OI signal effects when compared to unlabeled controls. ICG labeling provided short term labeling with rapid excretion of the label from the body while ferumoxides labeling allowed for cell tracking over several weeks.
  • Significance: The derived data allowed to establish and optimize hESC labeling with FDA approved contrast agents for a non-invasive depiction of the labeled cells with MR and OI imaging techniques. Our method is in principle readily applicable for monitoring of hESC -based therapies in patients and allows for direct correlations between the presence and distribution of hESC-derived cells in the target organ and functional improvements. The results of this study will be the basis for a variety of in vivo applications and associated further grant applications.

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