Long-term culture of human pancreatic slices as a model to study real-time islet regeneration.

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.post-print3.907 K

Single-cell resolution analysis of the human pancreatic ductal progenitor cell niche.

We have described multipotent progenitor-like cells within the major pancreatic ducts (MPDs) of the human pancreas. They express PDX1, its surrogate surface marker P2RY1, and the bone morphogenetic protein (BMP) receptor 1A (BMPR1A)/activin-like kinase 3 (ALK3), but not carbonic anhydrase II (CAII). Here we report the single-cell RNA sequencing (scRNA-seq) of ALK3bright+-sorted ductal cells, a fraction that harbors BMP-responsive progenitor-like cells. Our analysis unveiled the existence of multiple subpopulations along two major axes, one that encompasses a gradient of ductal cell differentiation stages, and another featuring cells with transitional phenotypes toward acinar tissue. A third potential ducto-endocrine axis is revealed upon integration of the ALK3bright+ dataset with a single-cell whole-pancreas transcriptome. When transplanted into immunodeficient mice, P2RY1+/ALK3bright+ populations (enriched in PDX1+/ALK3+/CAII− cells) differentiate into all pancreatic lineages, including functional β-cells. This process is accelerated when hosts are treated systemically with an ALK3 agonist. We found PDX1+/ALK3+/CAII− progenitor-like cells in the MPDs of types 1 and 2 diabetes donors, regardless of the duration of the disease. Our findings open the door to the pharmacological activation of progenitor cells in situ.post-print3.184 K

TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero

Pancreatic Stem Cells

The aim of Pancreatic Stem Cells is to provide a broad overview of an intriguing model of organogenesis (the development of the pancreas) from the perspective of stem cell research. The tangible prospect of devising effective cell therapies for type I diabetes –a disease thus far considered incurable– has stoked a widespread interest in harnessing our knowledge on the basic biology behind the development of this organ. This text is conceived as an all-encompassing guide to explore the many aspects of "regenerative therapies" for the endocrine pancreas, from adult and embryonic stem cells to endogenous regeneration and transdifferentiation. All these themes are built upon an up-to-date discussion on pancreatic ontogeny, which provides a solid background for both basic scientists and health professionals

Stem Cell Differentiation: General Approaches

Regardless of the cell type used as a building block for differentiation/transdifferentiation into pancreatic cells, there are only a few strategies that can be used to modify, both in vitro and in vivo, their fate and behavior. Conventional approaches are based on the addition of chemical soluble agents to the culture medium (signal-driven strategies), in an attempt to mimic the complex symphony of differentiation/specification factors that drive the process in vivo. Extracellular matrices and cell growth substrates may help increase the overall efficiency of these methods. Alternatively, external signaling can be bypassed by means of adding constitutively activated copies of key transcription factors or – more recently – cell-permeable proteins. The rationale of in vivo differentiation is that only the recipient’s body can provide developing cells with the adequate microenvironment to support terminal maturation

Pancreatic Development

Pancreatic development is arguably the best-studied example of organogenesis. Both gain-of-function and loss-of-function studies conducted in mice over the last decade have contributed to our understanding of a basic “genetic roadmap” of pancreatic – and particularly endocrine – development. Here we review this knowledge from the onset of the pancreatic program in the foregut epithelium (with the expression of the critical regulators Pdx1 and Ptf1a) to the specification of ductal, exocrine, and endocrine cell types. A special emphasis is placed on the development of endocrine beta cells, which are destroyed in type I diabetes and therefore constitute the endpoint of many stem cell differentiation protocols

Remaining Challenges and Clinical Perspectives

Unlike other potential targets of future stem cell approaches, there is already a current cell therapy for the treatment of type I diabetes. Indeed, islet transplantation has proven successful in inducing insulin independence for at least 1 year after the procedure. Progress in this discipline during the past 20 years has paved the way for stem cell-based therapies. Here we review the current state of the art of islet transplantation and examine the challenges that need to be addressed before a transition is made to stem cell-derived insulin-producing cells, with particular emphasis on the immunological aspects (rejection and autoimmunity) of type I diabetes

Pancreatic Reprogramming

Hidden behind the hype of prospective stem cell-based approaches to treat human disease, reprogramming techniques have finally entered the landscape of regenerative medicine and are quickly becoming one of the most exciting and powerful weapons in the field. In the context of pancreatic regeneration, the reprogramming of non-endocrine adult tissues to cells with phenotypes resembling to those of the hormone-producing cells of the islets of Langerhans is a fertile and dynamic area of research. Here we analyze two of the most studied sources of reprogrammable cells, namely the liver and the acinar compartment of the pancreas. Several groups have now established that the ectopic expression of master pancreatic regulators such as Pdx1, MafA, Ngn3, or BETA2/NeuroD can result in variable degrees of reprogramming toward pancreatic endocrine fates, leading to insulin production in vitro, and reversal of hyperglycemia in vivo. The state of the art and clinical prospects of these novel approaches are discussed in the following chapter

Adult Stem Cells and Pancreatic Differentiation

Adult stem cells are found in most tissues, where they are thought to participate in natural turnover and regeneration. Under defined conditions, some of these cells can also be significantly expanded and differentiated along specific lineages. This chapter is focused on mesenchymal stem cells (MSCs), which can be isolated from virtually every organ of the human body. While MSCs have a well-proven potential to give rise to connective tissues (e.g., bone, cartilage, fat, etc.), their ability to differentiate into endodermal cell types (and particularly insulin-producing beta cells) is not as clear. However, there is significant evidence that specific treatments may induce insulin expression, even if doubts remain about the true nature of the end product. At any rate, MSCs have other extraordinary features that go beyond their differentiation potential, as they may provide other cells with appropriate engraftment/differentiation niches