CHEMICAL REPROGRAMMING OF ASTROCYTES INTO FUNCTIONAL NEURONS FOR CNS REPAIR
Open Access
- Author:
- Zhang, Lei
- Graduate Program:
- Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 05, 2016
- Committee Members:
- Lei Zhang, Dissertation Advisor/Co-Advisor
- Keywords:
- Small molecule
Neuron
Astrocytes
Reprogramming
CNS repair - Abstract:
- The mammalian central nervous system (CNS) possesses very limited self-repair capability: very few newborn neurons are generated during adulthood. Regeneration of neurons in the CNS when injured or under pathological conditions remains a major challenge for functional recovery. Current efforts largely focus on cell replacement therapy with exogenous cells derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) to generate neurons (Sahni and Kessler, 2010; Takahashi et al., 2007; Takahashi and Yamanaka, 2006). In spite of the great promise, cell transplantation approaches face significant hurdles, such as poor survival rate, immunorejection, tumorigenesis and differentiation uncertainty (Lee et al., 2013; Lukovic et al., 2014). Glial cells represent a large reservoir for generating neurons locally. In response to CNS injury, glial cells (e.g., astrocytes, NG2 cells and microglia) are activated to proliferate and become hypertrophic to occupy the injured CNS area, thus limiting the spreading of injury in the acute stage (Pekny and Nilsson, 2005; Robel et al., 2011; Sofroniew and Vinters, 2010). On the other hand, long-term occupancy of the injury sites by reactive glial cells will result in the secretion of neuroinhibitory factors that prevent neuronal growth, eventually forming glial scars inside the CNS (Sofroniew and Vinters, 2010). Reactive glial cells have been widely reported after brain injury, spinal cord injury and neurodegenerative disorders, such as Alzheimer’s disease (AD) (Burda and Sofroniew, 2014; Gwak et al., 2012; Pekny and Nilsson, 2005; Sofroniew and Vinters, 2010; Verkhratsky et al., 2012). Recent studies, including our own, have demonstrated that astroglial cells can be directly converted into functional neurons in vitro (Guo et al., 2014; Heinrich et al., 2010; Zhang et al., 2015) and in vivo (Grande et al., 2013; Guo et al., 2014; Heinrich et al., 2010; Liu et al., 2013; Torper et al., 2013) by ectopic overexpression of neural transcription factors (TFs). So far, conversion of glial cells into neurons has been largely achieved using viral-based expression of TFs, but clinical applications might be hampered due to complex brain surgery and genetic alteration. Here we report a novel technology, chemical reprogramming that uses defined small molecules to reprogram cultured human astrocytes into functional neurons with high efficiency. Chemically converted human neurons can survive in long-term culture and in the mouse brain. Moreover, they form elaborate neuronal networks in culture and can integrate into mouse neural circuits. We further examined the mechanisms underlying small molecule-induced glia-to-neuron conversion. Our results suggest that human neurons can be directly generated by conversion of human astrocytes without a transient stem cell stage. Intriguingly, epigenetic silencing of glial genes and transcriptional activation of neural TFs, such as NEUROD1 and NGN2, are involved in chemical reprogramming. In addition, we evaluated the functional role of each individual small molecule included in the cocktail and identified the core molecules that are essential for highly efficient glia-to-neuron reprogramming. Towards future therapeutic applications, we also tested the effect of a small-molecule cocktail on neurogenesis in vivo. Interestingly, the small molecules injected into mouse brains induced stem cell properties in cortical and striatal astrocytes. Moreover, adult neurogenesis was significantly enhanced by local injection of small molecules in adult mouse hippocampus. In conclusion, our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons for CNS repair. Our chemical reprogramming method may potentially be translated into clinical therapy to help patients suffering from CNS disorders.