Interplay of Extrinsic and Intrinsic Cues in Cell-Fate Decisions

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Doctor of Philosophy (PhD)
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Stem Cell Engineering
Synthetic Biology
Systems Biology
Cell Differentiation
Nonlinear Dynamics
Biomedical Engineering and Bioengineering
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A cell’s decision making process is coordinated by dynamic interplay between its extracellular environment and its intracellular milieu. For example, during stem cell differentiation, fate decisions are believed to be ultimately controlled by differential expression of lineage-specific transcription factors, but cytokine receptor signals also play a crucial instructive role in addition to providing permissive proliferation and survival cues. Here, we present a minimal computational framework that integrates the intrinsic and extrinsic regulatory elements implicated in the commitment of hematopoietic progenitor cells to mature red blood cells (Chapter 2). Our model highlights the importance of bidirectional interactions between cytokine receptors and transcription factors in conferring properties such as ultrasensitivity and bistability to differentiating cells. These system-level properties can induce a switch-like characteristic during differentiation and provide robustness to the mature state. We then experimentally test predictions from this lineage commitment model in a model system for studying erythropoiesis (Chapter 3). Our experiments show that hemoglobin synthesis is highly switch-like in response to cytokine and cells undergoing lineage commitment possess memory of earlier cytokine signals. We show that erythrocyte-specific receptor and transcription factor are indeed synchronously co-upregulated and the heterogeneity in their expression is positively correlated during differentiation, confirming the presence of autofeedback and receptor-mediated positive feedback loops. To evaluate the possibility of employing this minimal topology as a synthetic “memory module” for cell engineering applications, we constructed this topology synthetically in Saccharomyces cerevisiae by integrating Arabidopsis thaliana signaling components with an endogenous yeast pathway (Chapter 4). Our experiments show that any graded and unimodal signaling pathway can be rationally rewired to achieve our desired topology and the resulting network immediately attains high ultrasensitivity and bimodality without tweaking. We further show that this topology can be tuned to regulate system dynamics such as activation/deactivation kinetics, signal amplitude, switching threshold and sensitivity. We conclude with a computational study to explore the generality of this interplay between extrinsic and intrinsic cues in hematopoiesis. We extend our minimal model analysis in Chapter 2 to examine the more complex fate decisions in bipotent and multipotent progenitors, particularly how these cells can make robust decisions in the presence of multiple extrinsic cues and intrinsic noise (Chapter 5). Our model provides support to both the instructive and stochastic theories of commitment: cell fates are ultimately driven by lineage-specific transcription factors, but cytokine signaling can strongly bias lineage commitment by regulating these inherently noisy cell-fate decisions with complex, pertinent behaviors such as ligand-mediated ultrasensitivity and robust multistability. The simulations further suggest that the kinetics of differentiation to a mature cell state can depend on the starting progenitor state as well as on the route of commitment that is chosen. Lastly, our model shows good agreement with lineage-specific receptor expression kinetics from microarray experiments and provides a computational framework that can integrate both classical and alternative commitment paths in hematopoiesis that have been observed experimentally.

Dr. Casim A. Sarkar
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