Research
Overview of the lab:
The Deans lab utilizes molecular biology, genetic engineering, and mathematical modeling to develop functional platforms of synthetic biology tailored for applications in stem cell biology, regenerative medicine, drug delivery, cell-based therapeutics, and disease monitoring. Our lab also interfaces synthetic biology and biomaterials to engineer bioinspired dynamic microenvironments to enhance stem cell proliferation in vitro.
Stem cells are important for tissue maintenance, regeneration, and repair. There is an increasing demand for stem cells because of the anticipated application in disease treatment, regeneration of damaged tissue, novel diagnostics, and pharmaceutical screens. Understanding the mechanisms of stem cell fate is essential to control their behavior for treating damaged and diseased tissue. Hematopoietic stem cells (HSCs) are adult stem cells that reside in the bone marrow and have the potential to self-renew and to differentiate into specialized blood and immune cells via a process called hematopoiesis (Fig. 1). Elucidating the mechanisms that regulate hematopoiesis will facilitate the understanding of normal hematopoiesis, pathologies involving blood cells, and enable hematopoiesis on demand for therapeutic applications. These cells are involved with controlling blood homeostasis, immune function, and have the ability to be activated in response to stress either by intrinsic or extrinsic stimuli, however, when the bone marrow is damaged or diseased, individuals develop blood diseases, autoimmune diseases, and/or cancer. Based upon the need for improved tools to study the mechanisms of stem cell fate decisions, our lab has three synergistic focus groups:
Figure 1: Overview of hematopoiesis. HSCs are multipotent stem cells that have the potential to differentiate into various precursor cells that become more specialized blood cells.
Expanding the genetic toolbox for mammalian cells to control the contribution of intrinsic cues on HSC proliferation and differentiation (synthetic biology)
The demand for stem cells will continue to rise because of their anticipated application in disease treatment, regeneration of damaged tissue, novel diagnostics, and pharmaceutical screens. However, before these goals can be realized, a better understanding of the mechanisms regulating their cell fate decisions is required for reliable reprogramming. Cells have the remarkable ability to continuously sense, integrate, and store relevant physiological and biological information over time. They integrate the many signals that surround them and execute complex cellular behaviors based on these inputs. These attributes can be harnessed and manipulated using advances in synthetic biology for creating enhanced diagnostic devices, and therapeutic cells that sense and respond with regulated doses of therapeutic biomolecules. An expanded mammalian toolbox would enable synthetic biology to address a broader range of questions in higher organisms. The tools built and used in synthetic biology are ideal for studying mechanisms involved in stem cell biology because they allow for the predicted and dynamic control of gene expression. Therefore, expanding the orthogonal genetic parts that function in mammalian cells will allow the construction of complex genetic circuits to probe, perturb, and regulate gene expression to study the underlying mechanisms of stem cell fate (Fig. 2).
Figure 2: Schematic of the LacQ genetic circuit. (A) The LacI repressor proteins are constitutively expressed (purple) and bind to the lac operator sites upstream of QF (orange) and GFP (green). This causes transcriptional repression of QF and GFP. (B) When IPTG is present, it binds to the LacI proteins and produces a conformational change in the repressor proteins. This causes the repressor proteins to no longer bind to the lac operator sites, allowing for the transcription of QF. Once QF proteins are produced, they bind to the QUAS binding sites and activate the transcription of GFP, resulting in a robust expression of GFP. From Fitzgerald, M., et al.
Control the intrinsic and extrinsic cues on HSC proliferation and differentiation (coupling synthetic biology with biomaterials)
Currently HSCs cannot be maintained and expanded in vitro because their proliferation and expansion requires a complex and dynamic microenvironment that has been difficult to engineer. This complex environment includes the coordinated interplay between the interactions of both intrinsic (transcription factor expression) and extrinsic (environmental) cues that contribute to HSC proliferation and their differentiation into lineage-committed cells. This interplay between intrinsic and extrinsic cues in hematopoiesis makes it difficult to study the mechanisms involved in their proliferation and their cell fate decisions. To study and control the intrinsic and extrinsic cues that take place during hematopoiesis, we are coupling synthetic biology with biomaterials (Fig. 3) to create dynamic biomimetic bone marrow microenvironments to be used for in vitro cell culturing systems.
Figure 3: Interfacing synthetic biology with biomaterials. Schematic of genetic circuit activation in PEG hydrogels. Cells harboring genetic circuits that are encapsulated in PEG (grey lines) hydrogels can be activated when the attached inducer molecules (orange stars) are released from the hydrogel. Various triggered release mechanisms have been shown to activate genetic circuits. Adapted from: Deans, T.L, et al. and Singh, A., et al.
Engineer cells monitor changes within their microenvironment while undergoing differentiation, and to deliver therapeutic biomolecules to sites of injury and disease (synthetic biology)
Utilizing tools in synthetic biology to tightly control gene expression in stem cells will not only enhance our understanding of stem cell fate decisions, but also enable directing stem cells into desired cell lineages. The ability to capture what a stem cell experiences in its native environment (i.e. changes in extracellular matrix (ECM), presence/absence of other cells, levels of proteins, etc.) as it differentiates would provide critical insight into how and when cell fate decisions are made. To accomplish this, we are engineering mammalian receptors that monitor extracellular events because this is an important step to reprogramming cells that sense and respond to their environment. Additionally, we are programming stem cells to function as novel delivery devices for damaged and diseased tissue.
Engineering bioinspired microenvironments
Recreating cellular microenvironments allows us to interrogate healthy and diseased tissue states, in addition to the progression from healthy to diseased states. We are specifically interested in engineering the complex microenvironment required to expand hematopoietic stem cells (HSCs) in vitro for transplantation procedures in patients with blood diseases, inherited blood disorders, autoimmune diseases, and/or cancer.