(7i) So This Engineer Walks into a Biology Lab: Regulating Cell Fate, Engineering Motor Neurons

>Building motor neurons to understand their unique functions and vulnerabilities

My interests lie at the intersection of neurobiology, systems biology, and synthetic biology, with my work primarily drawing on aspects of control theory and molecular biology. As a chemical engineer working in biological systems for the last twelve years, I have a unique blend of wet lab and computational expertise needed to identify, engineer, and reprogram both natural and synthetic biological networks. Through my postdoctoral training in stem cell biology and neurobiology, I have developed a passion for elucidating the molecular and cellular properties of motor neurons (MNs) that make them uniquely vulnerable to disease. I plan to establish a lab focused on analyzing native networks, constructing synthetic circuits, and designing molecular parts to engineer and understand the complex cellular behaviors that govern MN biology.

>Research accomplishments

As a graduate student at Caltech in Dr. Christina Smolke’s lab, I trained in molecular systems biology and synthetic biology, integrating non-coding RNA regulatory devices into synthetic circuits. By identifying pathway regulators of MAPK signaling, I constructed synthetic gene circuits and used these regulators to develop a class of genetic control systems called molecular network diverters. By interfacing these molecular network diverters with the native MAPK signaling pathway, I could temporally and spatially control cellular decision-making events (Galloway, K.E. et al. Science 2013). Beyond controlling cell fate, the design and construction of molecular network diverters allowed us to explore fundamental principles of integrating native and synthetic circuitry. For example, integrated negative regulators can buffer a system against noise amplification mediated through positive-feedback loops by providing a resistance to amplification. By constructing synthetic circuitry, I can illuminate paradigms in biological control and apply those principles to enhance cellular programming.

To diversify my experience and bring molecular systems biology principles to bear on questions of human health, I chose to pursue a position in stem cell biology and neurobiology. As a postdoc at USC in Dr. Justin Ichida’s lab, my work has focused on elucidating and overcoming the reprogramming roadblocks that hinder the robust generation of mature cell types. Accurately modeling neurological disorders with in vitro cellular models relies on robust, reliable methods to efficiently generate the distinct neural subpopulations affected by the disease. When I began my project, the conversion process was extremely inefficient, requiring large-scale efforts to generate only a few hundred cells. Moreover, the central mechanistic rules for direct lineage conversion were undefined. Today, as a direct result of my work to improve the reprogramming process, I can robustly generate thousands of cells with signatures of enhanced maturity. I now have the scale and quality needed to perform important biochemical assays on MNs including RNAseq and ChIPseq.

In addition to improving the reprogramming process, my work uncovers a previously unrecognized explanation for why only rare cells undergo transcription factor-mediated reprogramming successfully. In short, only a subset of cells have the transcriptional capacity to process the demand of transcription factor-mediated reprogramming. Reprogramming requires high levels of transcriptional machinery to mediate the extensive shift in transcriptional programs. By modulating the expression and activity of topoisomerases—vital components of the cell’s transcriptional machinery—I can expand transcriptional capacity, enhance reprogramming efficiency, and generate more mature cellular behaviors. I have shown that this is not only true for neurons, but is generalizable to other cell lineages in both mice and humans.

>Research directions

MNs provide a well-defined neuronal cell type with established molecular markers of identity and characterized sets of behaviors. Additionally, mouse models provide access to primary embryonic stage MNs for transcriptional and epigenetic reference. However, the inaccessibility of MNs in adult vertebrates hinders the isolation of mature cell types in significant numbers, preventing the characterization of fundamental cellular properties including epigenetic regulation and dynamic signaling behaviors. Due to these limitations, the mechanisms and pathology of MN diseases (e.g. Amyotrophic Lateral Sclerosis (ALS), a.k.a. Lou Gehrig’s disease) remain poorly understood with limited therapeutic options. Engineered human MNs provide a model for examining the unique functions and vulnerabilities that may contribute to the disease-specific sensitivity of in vivo MNs. The enhanced reprogramming methods that I have developed allow me to generate a diversity of MN subtypes in sufficient numbers to explore the behaviors and unexamined regulatory properties of MNs and their subtypes.

Neurodegenerative diseases, such as ALS, affect highly specific cell types in the central nervous system. ALS selectively destroys spinal MNs, which eventually leads to systemic paralysis and death. Given the human-specific context of neurodegenerative diseases and inaccessibility of primary cells, in vitro-derived MNs provide a model for interrogating a range of fundamental MN properties. The reliance on “disease-in-a-dish” models of neurodegenerative diseases is predicated on the idea that the affected cell type will recapitulate the cell-autonomous features of the disease. For ALS, we have only begun to scratch the surface of affected cells. Currently, the MNs used for disease modeling represent one of the least and latest affected subtypes of MNs in the ALS disease course. While these iMNs appear vulnerable to disease stimuli in vitro, their in vivo resilience suggests that they may misdirect efforts to identify therapeutic compounds. By generating the subtypes most acutely affected during the disease course, I will illuminate pathways that may explain the unique vulnerability of MN subtypes in ALS (Focus 1).

The human-specific context of ALS, inaccessibility of primary MNs, and limited number of in vitro-derived cells has limited our ability to probe the molecular determinants of the disease for most patients. Despite the accumulating whole genome sequencing data, the genetic determinants of the disease remain elusive. 80% of ALS patients do not have known causative genetic mutations in the examined coding regions, suggesting that mutation or dysfunction of the regulatory elements (e.g. enhancers) might underlie the majority of ALS incidences. Today, because of my work to improve the efficiency of generating in vitro-derived cells, I routinely generate sufficient quantities of motor neurons to construct enhancer maps from both mouse and human cells and have begun construction of a MN atlas of regulatory regions. Construction of MN enhancer maps from healthy and patient cells will enable me to identify dysregulated enhancers and enhancer variants that contribute to ALS pathology for patients with undefined genetic causes of the disease (Focus 2).

>Funding

Continuous independent funding as postdoctoral fellow with acquisition of additional mini-grants

NIH Ruth L. Kirschstein NRSA Postdoctoral Fellowship (Fall 2015 – Fall 2018)

California Institute of Regenerative Medicine Postdoctoral Fellowship (Fall 2013 – Fall 2015)

Doerr USC Stem Cell Challenge Award $10,000 project (Winter 2017)

Fluidigm USC Single Cell Project Grant $9,000 in reagents and materials (Summer 2016)

>Publications

Babos, K*., Galloway, K.E*., Kisler, K., and Ichida, J.I. Transcriptional capacity limits reprogramming. (In preparation). *These authors contributed equally to this work.

Galloway, K.E., Babos, K., and Ichida, J.I. Enhancing in vitro disease models of ALS through precise motor neuron subtype engineering. (In preparation).

Galloway, K.E*., Yu, H. *., Segil, N. I., and Ichida, J.I. Building a motor neuron enhancer map using ATM-ChIPseq. (In preparation). *These authors contributed equally to this work.

Galloway, K.E. and Ichida, J.I. Modeling neurodegenerative diseases and neurodevelopmental disorders with reprogrammed cells. Stem Cells, Tissue Engineering and Regenerative Medicine. D.A. Warburton, Ed. (World Scientific, New Jersey, 2015).

Franco, E., and Galloway, K.E. Feedback loops in biological networks. Computational Methods in Synthetic Biology. M. A. Marchisio, Ed. (Springer New York, 2015), vol. 1244, pp. 193-214.

Galloway K.E., Franco, E., and Smolke, C.D. Dynamically reshaping signaling networks to program cell fate via genetic controllers. Science. 2013. 341:1235005.

Chen, Y.Y*, Galloway, K.E.*, and Smolke, C.D. Synthetic biology: advancing biological frontiers by building synthetic systems. Genome Biology. 2012. 13:240. *These authors contributed equally to this work.

Kostal, J., Mulchandani, A., Gropp, K.E., and Chen, W. A. Temperature Responsive Biopolymer for Mercury Remediation. Environmental Science & Technology. 2003. 37, 4457-4462.

Teaching Interests:

My comprehensive chemical engineering education from UC Berkeley and Caltech makes me comfortable teaching most classes in the ChE curriculum (kinetics, transport, and thermodynamics) at both undergraduate and graduate levels. I’m also very familiar with control theory and molecular and cellular biology. At Caltech, I took several courses on molecular design and engineering, control theory, systems biology, and synthetic biology. The problem-solving skills I acquired as a chemical engineer have set me up for success in elucidating principles of molecular systems biology. Thus, I am enthusiastic to teach chemical engineering to the next generation of students.

In addition to serving as a teaching assistant for two classes at Caltech, I taught the freshman chemistry lab at Harvey Mudd College (HMC) in spring 2013 before beginning my postdoc. Over my four years as a postdoc, I have directly mentored 12 students (5 graduate, 5 undergraduate, 2 high school) which has motivated me to develop strategies for training my students 1) to be effective in lab and 2) to develop their research and presentation skills.