Use of Human induced-pluripotent Stem Cells to Understand Molecular Mechanisms of Autism
Open Access
- Author:
- Tang, Xin
- Graduate Program:
- Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 21, 2014
- Committee Members:
- Gong Chen, Dissertation Advisor/Co-Advisor
Douglas Cavener, Committee Chair/Co-Chair
Bernhard Luscher, Committee Member
Melissa Rolls, Committee Member
Yingwei Mao, Committee Member - Keywords:
- Human iPSC
astrocyte
neuron
functional maturation
synapse
KCC2
GABA functional switch
MeCP2
REST
Rett syndrome
Autism - Abstract:
- Despite having diverse capabilties, the human brain has limited ability in regenerating itself. This drawback hinders brain repair process in brain injury or neurodegenerative conditions, and imposes serious limitations to research efforts using human brain cells. In a major paradigm-shift, recent advances in stem cell biology have empowered scientists with the ability to generate a large quantity of human brain cells from stem cells. These technological breakthroughs not only offer great hope for regenerative medicine, but also make it possible to systematically study brain functions utilizing human brain cells. In order to realize the full potential of stem cell technology in studying the human brain, it is critical to derive various human brain cell types in significant amount, at the maturation stages similar to the endogenous human brain cells. Different protocols have been used to differentiate human iPS cells (induced-Pluripotent Stem Cells) into neurons for disease modeling and tranplantation, but their functional maturation process has varied greatly among different studies. In this dissertation, we demonstrate that laminin, a commonly used substrate for iPSC cultures, was inefficient in promoting full functional maturation of hiPSC-derived neurons. In contrast, astroglial substrate greatly accelerated the neurodevelopmental processes of hiPSC-derived neurons, which highlight a critical role of astrocytes in promoting neural differentiation and functional maturation of human neurons derived from hiPSCs. Moreover, our data provide a reliable protocol to generate functionally mature human neurons from stem cells, and present a thorough developmental timeline of normal human iPSC-derived neurons, as benchmarks to detect disease-relevant phenotypes and assess the efficiency of therapeutic agents. We next employed our glia-NPC (Neuroprogenitor Cell) co-culture system to study the molecular mechanisms of Rett syndrome, a severe form of autism spectrum disorder mainly caused by dysfunction of a single gene MeCP2 (Methyl-CpG binding Protein 2) located on the X-chromosome (Chahrour and Zoghbi, 2007). Rett patients develop relatively normally within 6-18 months after birth but then display developmental regression with impaired motor function, cognitive deficit, and even premature death (Chahrour and Zoghbi, 2007). Previous studies have demonstrated that MeCP2 regulates global gene transcription, and have identified a variety of downstream signaling molecules in neurons (Zoghbi and Bear, 2012; Ebert et al., 2013a). However, why Rett patients show a delayed developmental regression after birth is still an unanswered question. In this dissertation, we demonstrated that KCC2 (Potassium Chloride Co-transporter 2), a K+-Cl- co-transporter that directly regulates both GABA and glutamatergic functions, may mediate the functional output of MeCP2 in governing the course of brain development. We discovered that human neurons differentiated from induced pluripotent stem cells (iPSCs) originated from Rett patient showed a significant deficit in KCC2 expression and a delayed GABA functional switch, which were rescued by IGF-1 (Insulin-like Growth Factor-1) treatment. Moreover, introduction of KCC2 into Rett human neurons lead to a rescue of Rett-relevant deficits in excitatory synapse function to levels comparable as using MeCP2 restoration. We have confirmed our major findings from human neuron experiments in parallel studies carried out in cultured mouse neurons utilizing gene expression manipulation methods. We further demonstrated that MeCP2 interacts with REST (RE1-Silencing Transcription factor) to regulate KCC2 expression. Together, we propose that reduction in KCC2 expression in Rett neurons may contribute to the developmental regression due to deficits in GABA functional switch and glutamatergic synapses. Therefore, increasing KCC2 expression level may be a novel therapeutic approach for the treatment of Rett syndrome and other autism spectrum disorders at large. In addition to our work on iPS cell-derived human neurons, we have developed a protocol to reproducibly generate human glial cells from iPS cells. Using the iPS cell-derived human glial cells and human astroglial cell lines, we have carefully analyzed the differences between human and mouse astrocytes in their gene expression and functional output. Interestingly, comparing to mouse glial cells, human glial cells strongly promote stem cell proliferation, but not their neuronal differentiation. When mouse neurons were cultured on human astrocytes, the formation of inhibitory synapses were greatly facilitated, while excitatory synapse formation was not changed. In addition, cultured human astrocytes secrete large amount of glutamate. Co-culture with neurons halted the secretion of glutamate from human astrocytes. Our functional experiments and parallel transcriptome-wide gene expression profiling highlight the inter-species difference between human and mouse astrocytes to aid future researches on understanding the unique functions of human brain, and will help develop effective therapies that target astrocytes to treat human brain diseases. In summary, our work has established a streamlined procedure to reliably generate human neurons and glial cells from iPS cells, characterized the molecular and functional differences between human and mouse astrocytes, and discovered that deficiency in KCC2 is a core symptom of Rett syndrome. Our novel understandings on the biology of human neurons and glial cells in health and disease conditions will provide critical insight into the inner workings of human brain, and lead to rational development of novel therapeutic strategies to effectively rectify disturbances thereof.