Organs-on-a-Chip.

Organs-on-chips, also known as "tissue chips" or microphysiological systems (MPS), are bioengineered microsystems capable of recreating aspects of human organ physiology and function and are in vitro tools with multiple applications in drug discovery and development. The ability to recapitulate human and animal tissues in physiologically relevant three-dimensional, multi-cellular environments allows applications in the drug development field, including; (1) use in assessing the safety and toxicity testing of potential therapeutics during early-stage preclinical drug development; (2) confirmation of drug/therapeutic efficacy in vitro; and (3) disease modeling of human tissues to recapitulate pathophysiology within specific subpopulations and even individuals, thereby advancing precision medicine efforts. This chapter will discuss the development and evolution of three-dimensional organ models over the past decade, and some of the opportunities offered by MPS technology that are not available through current standard two-dimensional cell cultures, or three-dimensional organoid systems. This chapter will outline future avenues of research in the MPS field, how cutting-edge biotechnology advances are expanding the applications for these systems, and discuss the current and future potential and challenges remaining for the field to address.

No evidence for mouse pancreatic beta-cell epithelial-mesenchymal transition in vitro.

We used cre/loxP-based genetic lineage tracing analysis to test a previously proposed hypothesis that in vitro cultured adult pancreatic beta-cells undergo epithelial-mesenchymal transition (EMT) to generate a highly proliferative, differentiation-competent population of mesenchymal islet "progenitor" cells. Our results in the mouse that are likely to be directly relevant to the human system show that adult mouse beta-cells do not undergo EMT in vitro and that the mesenchymal cells that arise in cultures of adult pancreas are not derived from beta-cells. We argue that these cells most likely originate from expansion of mesenchymal cells integral to the heterogeneous pancreatic islet preparations. As such, these mesenchymal "progenitors" might not represent the best possible source for generation of physiologically competent beta-cells for treatment of diabetes.

Autotherapies: Enhancing Endogenous Healing and Regeneration.

The promise of tissue engineering and regenerative medicine to reduce the burden of disease and improve quality of life are widely acknowledged. Traditional tissue engineering and regenerative medicine approaches rely on generation of tissue constructs in vitro for subsequent transplantation or injection of exogenously manipulated cells into a host. While promising, few such therapies have succeeded in clinical practice. Here, we propose that recent advances in stem cell and developmental biology, immunology, bioengineering, and material sciences, position us to develop a new generation of in vivo regenerative medicine therapies, which we term autotherapies. Autotherapies are strategies based on optimizing endogenous tissue responses and capitalizing on manipulation of stem cell niches and endogenous tissue microenvironments to enhance tissue healing and regeneration.

Commentary: engineering of tissue healing and regeneration.

Regenerative medicine aims to restore homeostasis of diseased tissues and organs. With time, engineered replacement tissue constructs will play an increasingly important role in achieving this goal. Equally important, however, will be the ability to resolve disease-associated inflammation and to optimize tissue regenerative capacity by specifically patterning the host tissue microenvironment. The tools of bioengineering are uniquely suited to meet these challenges. Here, the candidate molecular and cellular targets for manipulating the host's inflammatory environment and tissue regenerative capacity are briefly discussed within the context of current and emerging bioengineering strategies. The objective is to draw the attention of basic scientists and engineers to the importance of regulating inflammation in achieving the goals of tissue engineering and regenerative medicine.

Generation of islet-like hormone-producing cells in vitro from adult human pancreas.

Transplantation of pancreatic islets can provide long-lasting insulin independence for diabetic patients, but the current islet supply is limited. Here we describe a new in vitro system that utilizes adult human pancreatic islet-enriched fractions to generate hormone-producing cells over 3-4 weeks of culture. By labeling proliferating cells with a retrovirus-expressing green fluorescent protein, we show that in this system hormone-producing cells are generated de novo. These hormone-producing cells aggregate to form islet-like cell clusters. The cell clusters, when tested in vitro, release insulin in response to glucose and other secretagogues. After transplantation into immunodeficient, nondiabetic mice, the islet-like cell clusters survive and release human insulin. We propose that this system will be useful as an experimental tool for investigating mechanisms for generating new islet cells from the postnatal pancreas, and for designing strategies to generate physiologically competent pancreatic islet cells ex vivo.

Small molecules convert fibroblasts into islet-like cells avoiding pluripotent state.

Obtaining large numbers of functional pancreatic islets via direct cellular reprogramming is an important clinically relevant goal. In a recent issue of Cell Stem Cell, Li et al. (2014) report a new step-wise protocol for generating islet cells from mouse embryonic fibroblasts using a combination of soluble molecules.

The defined combination of growth factors controls generation of long-term-replicating islet progenitor-like cells from cultures of adult mouse pancreas.

Application of pancreatic islet transplantation to treatment of diabetes is severely hampered by the inadequate islet supply. This problem could in principle be overcome by generating islet cells from adult pancreas in vitro. Although it is possible to obtain replicating cells from cultures of adult pancreas, these cells, when significantly expanded in vitro, progressively lose pancreatic-specific gene expression, including that of a "master" homeobox transcription factor Pdx1. Here we show for the first time that long-term proliferating islet progenitor-like cells (IPLCs) stably expressing high levels of Pdx1 and other genes that control early pancreatic development can be derived from cultures of adult mouse pancreas under serum-free defined culture conditions. Moreover, we show that cells derived thus can be maintained in continuous culture for at least 6 months without any substantial loss of early pancreatic phenotype. Upon growth factor withdrawal, the IPLCs organize into cell clusters and undergo endocrine differentiation of various degrees in a line-dependent manner. We propose that our experimental strategy will provide a framework for developing efficient approaches for ex vivo expansion of islet cell mass.

Adult pancreas generates multipotent stem cells and pancreatic and nonpancreatic progeny.

Strategies designed to produce functional cells from stem cells or from mature cells hold great promise for treatment of different cell-degenerative diseases. Type 1 and type 2 diabetes are examples of such diseases. Although different in origin, both involve inadequate cell mass of insulin-producing beta cells, the most abundant cell type of pancreatic islets of Langerhans. Practical realization of such strategies is highly dependent on the elucidation of physiological mechanisms responsible for generation of new beta cells in the pancreas, which at this time are poorly defined. The in vitro differentiation systems allowing generation of new beta cells provide a valuable experimental tool for studying these mechanisms. Few such systems are currently available. In this work, we present an in vitro differentiation system, derived from adult mouse pancreas, capable of generating insulin-producing beta-like cells, which self-organize into islet-like cell clusters (ILCCs) during the course of the culture. Surprisingly, we found that along with the ILCCs, multiple cell types with phenotypic characteristics of embryonic central nervous system and neural crest are also generated. Moreover, several embryonic stem cell-specific genes are induced during the course of these cultures. These results suggest that the adult pancreas may contain cells competent to give rise to new endocrine and neural cells.

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease.

Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.