Single Cell Analysis of Retrotransposition and Genetic Mosaicism during Stem Cell-based Neurogenesis
Basic Biology III
Bona fide human neurons can now be studied directly in ways that have never before been possible. This is due to the ability of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) to differentiate into any cell in the body. However, it is critical that we learn both the similarites and differences between these two types of stem cells with regard to how well hESC-derived and hiPSC-derived human neurons resemble true human neurons. In addition, patient-derived hiPSCs provide a new avenue to help understand how human neurons give rise to neuropsychiatric disorders, such as schizophrenia. Whereas most healthy organs are made up of cells with identical genomes, it has recently become clear that the human brain is composed of many neurons that have unique variants of that individual's genome. Genomic diversity among an individual's neuronal genomes is brought about, in part, through the action of L1 retrotransposons. L1 retrotransposons are "jumping genes" that copy-and-paste themselves into new places in different cells through a process known as retrotransposition. Some level of L1 retrotransposition is normal and may contribute to individuality, particularly between identical twins. However, too much retrotransposition has been associated with neurological disorders. We propose to study L1 retrotransposition in hESC- and hiPSC-derived neurons from different individuals alongside hiPSC-derived neurons from schizophrenia patients. The proposed research will determine the extent to which L1 retrotransposition in stem cell-derived neurons recapitulates L1 activity during normal human development, and examine roles for retrotransposition in bringing about neural connectivity abnormalities observed a newly developed hiPSC-based model of schizophrenia.
Statement of Benefit to California:
Neuropsychiatric diseases affect millions of Californians directly, and untold more indirectly through the additional stess placed on a patient's family and loved ones. In addition, neuropsychiatric patients are at increased risk of homelessness and substance abuse that places additional burden broadly on all Californians. In passing Proposition 71, Californians asked California's scientists to advance stem cell research in meaningful ways. Neurons derived from stem cell are poised to be game-changers toward understanding and treating neuropsychiatric disease; thus, the proposed research promises to benefit all Californians by advancing our general understanding of stem cell-derived neurons, and by specifically investigating the neurodevelopmental basis of schizophrenia.
Project Synopsis: This proposal is focused on the role of L1 retrotransposons, so-called “jumping genes”, in human stem cell biology and schizophrenia. L1 retrotransposition has been shown to alter the genome both during the culture of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs, respectively) as well as during normal mouse brain development. This process could affect the genomic stability of cultured pluripotent stem cells and also play a role in human neurological diseases, such as schizophrenia. The applicant proposes to use single-cell genomic analysis to study these issues in three Specific Aims: (1) to compare L1 retrotransposition in different types and subtypes of pluripotent and neural progenitor cells; (2) to compare L1 retrotransposition in hiPSCs derived from schizophrenic patients and healthy controls; and (3) to determine whether L1 retrotransposition causes other genomic changes during the differentiation of pluripotent cells into neurons. Significance and Innovation: - Reviewers noted that gene copy number variation (CNV) and a correlating increase in L1 retrotransposition have both been described in hiPSCs from Rett syndrome and schizophrenic patients. They agreed that understanding these genomic changes in pluripotent cells and neural progenitors would be a significant advance. - It may be too early to conclude that the project would have a major impact on potential applications in regenerative medicine. - The proposed single-cell genomic analysis technology is both innovative and cutting-edge. - The rationale for the project is logical and scientifically sound. Feasibility and Experimental Design: - Reviewers appreciated the strong preliminary data supporting the project’s feasibility but raised some concerns about the experimental design. - One major concern is that the healthy controls proposed for Aim 2 are fibroblast donors unrelated to the schizophrenic patient donors. Reviewers noted that these samples may differ significantly in their CNV susceptibility and suggested that healthy family members of the schizophrenic patients would be important controls. The same concern applied to the proposed post-mortem controls. - The technology development proposed in Aim 3 to improve throughput may be required to generate meaningful data from Aims 1 and 2. It would have been helpful to have these techniques in place at the outset. Principal Investigator (PI) and Research Team: - Significant concerns were raised about the qualifications of the principal investigator (PI) due to limited evidence of success as an independent investigator. Reviewers stressed that the PI’s credibility on this topic will be more convincing upon publication of the submitted/in preparation manuscripts. - The PI only lists five publications since 2001, none of which are senior author papers. Reviewers were uncertain of the nature of the PI’s position as Staff Scientist and whether it is independent of a senior collaborating investigator. They would have appreciated greater clarification about the personnel roles on the project and the independence of the PI. Responsiveness to the RFA: - This project is responsive to the RFA, as it is focuses on basic mechanisms underlying the developmental potential of human stem cells as well as disease-in-a-dish modeling.
- Stephen L Minger
- Chad Cowan