Systems Biology: Living cell arrays

Transcription factors (TFs) are targeted for the quantification of activity as they are powerful regulators of cell differentiation, evidenced by the ability of a few TFs to reprogram fibroblasts to iPS cells. Furthermore, TFs are downstream targets of signaling pathways and large-scale TF quantification can reflect the activity throughout the intracellular signaling network. The large scale, dynamic, quantification of transcription factor (TF) activity is achieved in an array of cells, with each position of the array reporting on a distinct TF. Bioluminescence imaging (BLI) from a multi-well plate allows for rapid large-scale analysis. Additionally, BLI is non-invasive and allows for repeated measurements over time scales of minutes to weeks. Importantly, TF activity assays can be performed for cells cultured in our designer niches, which consist of tunable hydrogels that allow cells to grow in 3D and recreate the structures normally observed in vivo. An important aspect of this research is the development of computational models to analysis the dynamic TF data.  This novel technology is central to the applications described below, including cancer biology,  immunomodulation, and cell fate decisions.

Researchers: Joseph Decker, Matthew Hall, Robert Kuo

Regenerative Medicine: Islet transplantation

IsletThe recent clinical successes using islet transplantation have demonstrated that cell replacement therapy has the potential to be a viable treatment for type 1 diabetes. Current clinical approaches deliver islets through the portal vein and subsequently reside within the sinusoids of the liver. This approach requires large numbers of islets due to limited survival or hypofunctionality of the islets following transplantation. A transformative approach to islet transplantation can be achieved by locally controlling the transplant microenvironment to proactively counter the challenges to islet survival, engraftment and long-term function while minimizing or eliminating systemic immunosuppression with tolerance induction or local immunomodulation. Enhanced survival and engraftment can reduce the islet mass needed, thus, dramatically increasing the availability to patients given the limited organ supply. Using an extravascular, extrahepatic approach, the IBMIR can be eliminated and the transplant microenvironment can be designed to provide for the metabolically demanding islets and to deliver biomolecular signals promoting islet function, engraftment and survival. Locally recapitulating the islets’ native environment can enhance survival and function, and may support the normal beta-cell turnover that is essential for long-term function. In addition, the transplant microenvironment can be designed to modulate the immune cell infiltration and activation associated with auto- and allo-immune attack, which may facilitate tolerance induction. Reducing or eliminating the systemic immunosuppressive load will lower the burden on patients and may significantly enhance the maintenance of insulin independence.

Researchers: Peter Rios, Jeffrey Liu, Tadas Kasputis, Dan Clough, Briana Dye, Richard Youngblood

Regenerative Medicine: Spinal Cord Regeneration

NerveSevere nerve damage, an injury that affects many thousands of patients each year, often results in loss of function, such as paralysis below the level of the injury. A crush injury to central nervous system (CNS) neurons can result in massive cell death and produce a cyst and glial scar that pose a formidable barrier to nerve regeneration. Pioneering studies done in the 1980’s identified that CNS neurons have the capacity for regeneration; however, the current strategies for augmenting nerve regeneration following injury have met with limited success. We have developed polymer scaffolds, termed bridges, that can be implanted into the spinal cord and aim to create a more permissive environment for regeneration. The bridge is highly porous to allow for cell infiltration that stabilizes the spinal cord, and has channels that orient and direct axonal elongation. Infiltrating cells can secrete factors that support some regeneration; however, the concentration of stimulatory factors is insufficient for regeneration and these cells may also produce inhibitor factors that limit regeneration. To further modulate the local environment, gene therapy vectors are delivered to either induce expression of stimulatory factors, or to block expression or degrade inhibitory factors. The delivery of gene therapy vectors provides a versatile tool to target a range of processes using a single delivery system. These bridges have the potential to create an environment that excludes the factors that inhibit regeneration and provides the factors that stimulate neurite outgrowth.

Researchers: Daniel Margul, Michael Skoumal, Courtney Dumont, Jonghyuck Park, Dom Smith, Jessica Chen

Regenerative Medicine: Oncofertility

FollicleThe treatment of cancer using chemotherapeutics can lead to loss of ovarian function and infertility. Ovarian tissue can be cryopreserved prior to cancer therapy, and we are working to harness the potential of this tissue to preserve the reproductive potential for these patients. Our strategies fall into two groups: i) In vitro culture systems for the maturation of follicles, or ii) the transplantation of ovarian tissue. The reproductive unit within the ovary is the follicle, which consists of an oocyte surrounded by one or two layers of granulosa cells. For in vitro culture, we employ a three-dimensional, engineered, synthetic stroma to examine follicle maturation and development in vitro. Oocyte maturation and granulosa cell development involve endocrine, paracrine, and autocrine-acting factors in addition to appropriate somatic-germ cell and somatic cell-matrix interactions. Previously used two-dimensional culture systems do not adequately retain the three-dimensional architecture. A synthetic scaffold can serve as a stroma that creates a cellular environment designed to provide the factors that stimulate maturation of the follicles, but lacks the factors found in the native stroma that inhibit maturation. Synthetic scaffolds can be created which maintain the appropriate size, shape and architecture of the tissue while providing the necessary signals to direct cellular responses. Alternatively, we are employing biomaterials and drug delivery as a mechanism to enhance the engraftment of transplanted follicles. In this approach, the follicles develop in vivo and the challenge lies with maximizing survival of the transplanted tissue.

Researchers: Ekaterina Kniazeva, Eiji Saito, Peter Rios

Regenerative and Tolerogenic Immunomodulation

ParticlesModulating the immune response with vaccines has been one of the greatest success stories in the history of medicine. New tools to modulate the response of specific immune cell populations could facilitate novel therapies for autoimmune disease and regenerative medicine, and we are developing distinctive capabilities aimed at promoting i) a less inflammatory and more regenerative phenotype for regenerative medicine, or ii) antigen-specific tolerance, which has applications for  autoimmune disorders, cell transplantation therapies, and protein therapeutics. In vivo reprogramming of immune cells: Strategies for controlling innate immunity (e.g., macrophage activation) are a crucial first step in promoting regeneration or inducing tolerance.  Infiltrating immune cells, such as macrophages, are a promising target for inhibiting the degenerative inflammatory response after SCI because they can be shifted from an inflammatory to a regenerative phenotype to downregulate inflammatory cytokine production, and upregulate scavenger receptor expression for enhanced clearing of inhibitory debris. Gene delivery from the biomaterial scaffolds leads to transfection of macrophages, which are investigating as a mechanism to reprogram these cells into assets for regeneration or cell engraftment. Technologies for induction of antigen specific tolerance: Tolerance research emerged from our success and collaborations with islet transplantation for type 1 diabetes (T1D), and has extended to autoimmune disorders and allogeneic cell transplantation. We have collaborated on the production of antigen-coupled nanoparticles for the induction of tolerance to inhibit the specific undesired immune response while not altering the remaining elements of the immune response. Nanoparticles are modified with specific antigens, which target the spleen following intravenous delivery and are internalized by ‘tolerogenic’ APCs to present the antigens without T cell activation thereby inducing tolerance rather than an immune response. Most strategies targeting the immune system focus on inducing a specific immune response (vaccines), whereas this system aims to do the opposite by inducing tolerance to specific antigens. Interestingly, tolerance induction was enhanced by islet delivery on scaffolds relative to hepatic transplantation, suggesting the local environment impacts tolerance. PLG nanoparticles modified with the antigen have been developed that are able to prevent the development of autoimmune disease. Particles without peptide, or modified with the control peptide did not prevent disease. Ongoing research is investigating the particle design that can lead to tolerance, as well as employing the cell array to investigate the effect of particles on the differentiation state of macrophages.

Researchers: Eiji Saito, Robert Kuo, Liam Casey, Ryan Pearson, Jeffrey Liu, Kevin Hughes

Cancer Initiation and Metastasis

The biology of tumors can no longer be perceived simply by enumerating the genetic mutations within cancer, and new technologies must be developed to encompass the contributions of the genetic mutations as well as the tumor microenvironment. We are investigating the transition from a normal to a pre-invasive phenotype, and a pre-invasive to invasive phenotype using our novel technologies, which includes designer hydrogels, co-cultures of mammary epithelial cells and stromal or immune cells, and the TF array. The analysis of TF activity during cancer progression is complemented by our mechanistic studies of drug action, which aims to identify the TF activity downstream of a number of pharmaceuticals in clinical trials. In the area of cancer metastases, we aim to detect metastases at the earliest stages, which could allow for life-preserving interventions. by implantation of a biomaterial scaffold that recruits metastatic cancer cells. Recruiting the cancer cells would initially function to reduce the burden of circulating tumor cells and prevent their colonization in other tissues. Through collaboration, we are investigating non-invasive detection systems to identify the time at which cancer cells have colonized the scaffold. Upon detection of the metastatic cells, the implant could be retrieved to analyze the biology of the cells as well as the stromal/immune environment. The development of an implant to recruit metastatic cells and a non-invasive technology to monitor growth could transform current clinical approaches to cancer treatment.

Researchers: Grace Bushnell, Rachel Dudek, Robert Oakes, Yining Zhang