Department researchers are pursuing engineering of tissue equivalents mimicking the native pancreas. Here a tissue section from mouse pancreas shows the insulin-producing cells fluorescing green against the rest of the pancreatic tissue (exocrine tissue) fluorescing red.
Tissue engineering, embryonic stem cells, adult stem cells, viral vectors, biochemical engineering
E. (Manolis) S. Tzanakakis, Assistant Professor
A major issue in the engineering of tissues and artificial organs is the source of the cellular component. The use of animal cells or cell lines is accompanied by a number of serious concerns including the possibility of adverse immune response and rejection, the formation of tumors and the transmission of pathogens to the recipient. To that end, stem cells may provide an alternative, abundant source of cells used for developing therapies for a wide spectrum of pathological conditions.
Generating insulin-producing cells from stem cells
Diabetes mellitus is a chronic, debilitating disease afflicting more than 6% of the US population and close to 200 million people worldwide while these numbers are expected to double by 2030. Type I (insulin-dependent) diabetes is characterized by the autoimmune destruction of insulin-producing pancreatic islets of Langerhans. Type 2 diabetes usually begins with as insulin resistance but as the need for insulin augments, the pancreas gradually loses its ability to produce insulin. Adult onset diabetes can be managed through careful diet, exercise and drug regimens including the administration of insulin in almost 30% of cases. Type I diabetes patients however, must have insulin delivered to survive. Conventional insulin therapy is an imperfect treatment for diabetes often failing to match insulin with actual blood glucose concentration. More importantly, insulin administration does not cure diabetes nor prevents serious long-term complications such as neuropathy, renal failure, stroke and heart attack.
To that end, cell therapy may provide an attractive, long-lasting alternative to insulin therapy. Recent advancements in the transplantation of donor islets have provided new hope for a cure but the demand by far outnumbers the supply of donor cells, creating the need for exploring alternative sources of graft material. Regeneration of beta-cells, engineering of non-beta-cells and differentiation of stem cells towards insulin-producing cells are key potential sources. Differentiated beta-cells have very limited proliferative capacity but ongoing research on the mechanisms underlying beta-cell cycle, may shed light on methods utilizing these cells for therapies. Attempts to engineer non-beta-cells for diabetes therapies have been met with limited success. For a cell-based insulin therapy to be useful, cells must exhibit specific functional properties: insulin gene expression, appropriate insulin post-translational modification and processing through a highly regulated secretory pathway, as well as glucose-sensitive insulin synthesis and release within physiological range. Furthermore, native islet structure appears to be important for proper cell function and glucose homeostasis.
Our laboratory is interested in the differentiation of stem cells towards insulin-producing cells. Because of their ability to self-renew, stem cells represent a promising solution to overcome the limited availability of donor islets cells. However, several issues must be addressed before stem cell-based therapies of diabetes become reality.
Current protocols for differentiation of embryonic stem cells and adult progenitor cells to pancreatic cell progeny are characterized by extremely low efficiency with only a minute fraction of the initial population exhibiting insulin-producing characteristics. We undertake efforts to establish differentiation methods drawing upon the current state of pancreas biology during development. Our culture strategies are based on mimicking conditions present in the milieu of the embryonic pancreas anlage. Using genetic and epigenetic methods, we seek to identify, a set of factors and conditions which may be sufficient to coax stem cells into acquiring a beta-like cell phenotype. Epigenetic methods are based on treatment of stem cells with pancreatic cell maturation factors while genetic methods rely on the delivery of genes encoding transcription factors, signaling molecules etc.
Engineering pancreatic islets
In human islet transplantation, the minimum number of islets required for insulin independence is about 0.6 x 106 for a 70-kg recipient, or 30-50% the normal islet number. Although production of sufficient amounts of cellular material is important, generation of three-dimensional islet-like clusters is also necessary considering the intimate link between endocrine islet cell function and structure. Beta cells organized in three-dimensional structures featuring similarities to the anatomical architecture of native islets exhibit enhanced beta-cell specific functions, synchronicity in response to stimuli, and prolonged cell viability in culture.
Our laboratory focuses on contriving a platform for engineering pancreatic islets. In line with our focus on stem cell differentiation towards beta-cells, we are exploring ways of culturing insulin-producing cells in large-scale to assemble into islet-like structures with defined characteristics. Dispersed beta cells have been shown to re-assemble into islet-like structures when cultured in low-adhesion substrata. Nevertheless, this type of culture suffers from a series of drawbacks including the difficulty in scaling up to meet larger production needs.
Islet-like structure assembly in static culture: (A) beta-cell monolayers, (B) beta-cell aggregates. Bar 100 micrometers
Stem cell expansion
Regardless of the intended end-point of differentiation, stem cells for use in clinical applications must be expanded in quantities far exceeding the scale of laboratory culture. Bearing in mind that stem cell self-renewal diminishes as differentiation progresses, the development of a scalable culture system for expansion of undifferentiated stem cells is highly desirable. Existing bioreactor technologies can provide the foundation upon which culture systems for the expansion of stem cells can be designed. However, this requires solid comprehension of the molecular mechanisms regulating the self-renewal of stem cells. Although various extrinsic factors and intracellular signaling cascades participating in stem cell proliferation are known, this picture of the self-renewal circuitry is incomplete. We seek to gain a deeper insight of the determinants required for preservation of undifferentiated state as well as to exploit current knowledge for instituting culture strategies aiming at large scale expansion of stem cells.
Rough schematic of self-renewal signaling in stem cells. Details about the active intermediates in various pathways as well as additional participating factors are the focus of ongoing research.
