Differentiation of Stem Cells into ‘Systems of Neurotransmitter' Phenotypes Related to Alzheimer’s and Huntington’s Diseases: Application of High Throughput Peptidomic Approaches with Mass Spectrometry

Funding Type: 
SEED Grant
Grant Number: 
RS1-00381
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Alzheimer’s disease (AD) and Huntington’s disease (HD) neurodegenerative diseases involve loss of neurons in certain brain regions. Replacement of neurons in these diseases by stem cells differentiated into functional neurotransmitter phenotypes has high potential to provide stem cell therapy for these diseases. The nervous system utilizes integrated actions of multiple neurotransmitters that mediate communication among neurons. It is, therefore, critical to identify factors that promote differentiation of human stem cells into systems of neurotransmitters present in normal neurons. However, most studies have examined functions of only one neurotransmitter at a time, rather the groups of transmitters that function together. It is,, therefore, important to assess factors that differentiate human stem cells into profiles of neurotransmitters that represent the normal neurons. Analyses of ‘systems of neurotransmitters’ requires advanced technology in mass spectrometry for determining profiles of neurotransmitters. With the strong and long-standing efforts of the Hook laboratory to investigate peptide neurotransmitters, we have implemented state-of-the-art peptidomic technology for high throughput LC-MS/MS mass spectrometry to identify neurotransmitters. This new state-of-the-art technology will be used for this project. Therefore, the goal of this project will be to evaluate agents that transform human stem cells into neurons containing the ‘system of neurotransmitters’ present in the normal condition of brain regions affected in AD and HD. The specific aims will (1) test growth factors and related differentiating agents to induce human stem cells into neurotransmitter phenotypes of brain regions affected in AD and HD, and (2) compare the ‘system of neurotransmitters’ in normal human hippocampus and striatum with that in differentiated stem cells to guide differentiating conditions to generate the normal ‘system of neurotransmitters’ in selected human brain regions. Such differentiated cells may provide benefit for cell therapy to improve the health of patients affected with neurodegenerative diseases.
Statement of Benefit to California: 
Numerous citizens in California are affected by devastating neurodegenerative diseases, including Alzheimer’s, Huntington’s, and Parkinsons’ disease. The unique opportunity for human stem cell therapy in California provides special research that can potentially improve health for California citizens affected by these neurodegenerative diseases. The focus of this project to utilize novel peptidomic technology to understand the systems of neurotransmitters utilized in brain function is critical to guiding research to differential human stem cells into functional brain neurons. Achievement of the goals of this project is key to providing California citizens with the hope of new medical treatments using stem cells to relieve affected patients of the detrimental neurodegeneration in brain diseases.
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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