Structural and Functional Imaging of Pancreatic Islets using Optical Coherence Microscopy

In diabetes, pancreatic beta-cells play a key role. These cells are organized within structures called islets of Langerhans inside the pancreas and produce insulin. Insulin is one of the main hormones contributing to glucose homeostasis, which is the hallmark of diabetes. In vivo imaging of beta-cells and their function are required for diagnosis purposes and assessment of new treatments. However, this task is challenging due to the localization of the pancreas deep inside the abdominal cavity and the small size and heterogeneous distribution of islets of Langerhans throughout the organ. Indeed, islets of Langerhans represent only 1-2% of the pancreas' total volume. All state-of-the-art medical imaging techniques such as MRI, SPECT or PET cannot resolve individual islets. Furthermore, they require the use of tracers to detect the beta-cells. Hence, the research for imaging islets of Langerhans, including the identification of specific beta-cell tracers is a highly active field of research. Indeed, no marker is currently available for clinical studies and diagnosis. For animal models, optical techniques offer a sufficient spatial resolution to image individual islets with a high specificity provided by fluorescent markers. Optical Coherence Microscopy (OCM) is a novel interferometric imaging technique measuring the back-scattered light from a sample to reveal its structure in depth. OCM provides an intrinsic contrast depending on the spatial variation of the index of refraction. A Fourier transform of the acquired spectrum extracts the whole depth structure, thereby requiring only scans in two dimensions to obtain a three-dimensional image with a fast acquisition time. In this thesis, we exploited the label-free capabilities and fast acquisition rate of OCM to study islets of Langerhans. We demonstrated that OCM signal is specific to the beta-cell volume due to the dominant scattering of the zinc-insulin crystalline structures inside the secretory granules. Besides structural information, OCM reveals the vascularization of pancreatic islets in situ. These advantages were exploited to image the progression of autoimmune diabetes and for the characterization of beta-cell tracers. The intrinsic contrast of OCM does not require genetically modified mice, nor the use of exogenous agents to image islets and their vascularization. OCM enhanced with a confocal fluorescence channel can assess beta-cell tracers labeled with a fluorophore in vitro and in vivo in wild type mice. As a proof of principle, we assessed the specificity of Cy5.5-exendin-3, an analogue of the glucagon-like peptide-1 receptor (GLP1R) for beta-cells. Our results confirmed the co-localization of the fluorescence-tagged tracer with the OCM islet signal. The high resolution of OCM serves as a pre-clinical optical platform to facilitate the initial tests aiming to determine the specificity of beta-cell tracers in vivo in mice. Finally, in order to perform non-invasive longitudinal studies, islets of Langerhans were transplanted into the anterior chamber of the eye. This transplantation model allowed us to follow individual islets over time in a spontaneous mouse model of type I diabetes. We demonstrated that alterations of the islet microvasculature accompany the progression of diabetes with a strong correlation between the degree of insulitis and the density of the vascular network.

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

The pancreas plays a central role in metabolism. The exocrine pancreas, composed of ductal and acinar cells, secretes and delivers digestive enzymes into the duodenum where they contribute to food digestion. The endocrine pancreas, constituted by the islets of Langerhans, secretes hormones in the blood to control glucose levels. In particular, insulin is the hormone produced by β-cells that instructs the peripheral tissues to uptake circulating glucose. Diabetes mellitus is a heterogeneous disease characterized by deregulated glucose homeostasis due to an impaired insulin function. The advancements in clinical practice have made of diabetes a chronic, instead of a lethal, disease; yet no definitive cure is available. Beta-cell transplantation offers promising results, especially for type 1 diabetes where β-cells are selectively eliminated by an autoimmune attack. Unfortunately its application suffers from the scarcity of transplantable pancreas/islets from cadaveric donors. Human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) would constitute unlimited sources of transplantable β-cells, but the available differentiation protocols are not yet optimal, especially with regards to the numbers and the functionality of the generated β-cells. It is anticipated that a stepwise protocol would succeed in generating mature β-cells in vitro if it is capable of fully mimicking the embryonic development of these cells. Many aspects of pancreatic organogenesis have been elucidated and could serve as a guide for driving the commitment to β-cells. Unfortunately, pancreatic progenitors in vitro do not behave as in the intricate context of an embryo. This severely limits both the understanding of pancreatic development at the single-cell level and our ability in manipulating the process. The aim of our work was to develop in vitro methodologies to sustain pancreatic progenitor culture and expansion and to establish conditions to manipulate their progeny. To this aim, we combined an informed approach based on developmental biology and an empirical one. We first observed that pancreatic epithelial explants from embryonic day 10.5 mice could be cultured in presence of Matrigel™ and exogenous FGFs even in the absence of the mesenchyme that normally surrounds the epithelium. Notably, the removal of mesenchyme did not affect the endocrine commitment pattern of pancreatic progenitors. We then defined culture conditions allowing for the expansion of dissociated embryonic pancreatic progenitors in a three-dimensional Matrigel™-based environment. Under these conditions, progenitors proliferated and self-organized to generate pancreatic organoids containing both progenitors and differentiated cells, mostly exocrine, after 7 days of culture. When grafted into recipient pancreatic explants, the cultured progenitors contributed to the three epithelial pancreatic lineages (ductal, endocrine, acinar), integrating seamlessly into the endogenous cellular network. Thus, cultured progenitors retained their potential to differentiate into endocrine cells, but expressed it only in the appropriate niche. Subsequent removal of single components from the culture system led to the identification of some essential requirements for the maintenance/expansion of dissociated pancreatic progenitors, such as a strong FGF signaling and the Rho-associated kinase (ROCK) inhibitor Y-27632. In addition to this, we observed that only small clusters of pancreatic progenitors, but not single cells, were able to generate pancreatic organoids, suggesting a pivotal role for intercellular signaling in progenitor maintenance and expansion. By manipulating the components of the medium, we could affect fate commitment. In particular, the removal of FGF1 increased the yield of endocrine cells generated in vitro. In our search for a full control over the in vitro niche, we explored chemically defined matrices to replace Matrigel™. We showed that pancreatic progenitors could be cultured on laminin-functionalized hydrogels, although less efficiently than in Matrigel™. Moreover, differentiation into exocrine and endocrine cells occurred spontaneously under these conditions. By comparing Matrigel™, hydrogel microwells and two-dimensional culture systems, we uncovered the importance of a tridimensional architecture for pancreatic progenitor maintenance. We showed that pancreatic progenitors lost their identity as they flattened onto 2D surfaces, whereas 3D culture systems maintained the epithelial character of pancreatic progenitors and allowed the acquisition of apical polarization. To conclude, our work provides the first detailed characterization of in vitro culture systems for expansion and manipulation of dissociated embryonic pancreatic progenitors. Further implementation will hopefully allow the establishment of a fully-defined in vitro niche for developing pancreas with potential fundamental implications for the expansion of ES-derived pancreatic progenitors and their differentiation into functional β-cells.

EPFL2011

Towards high resolution optical imaging of beta cells in vivo

Joan Goulley, Anne Grapin-Botton, Theo Lasser, Erica Martin-Williams, Martin Villiger

Endocrine beta cells produce and release insulin in order to tightly regulate glucose homeostasis and prevent metabolic pathologies such as Diabetes Mellitus. Optical imaging has contributed greatly to our current understanding of beta cell structure and function. In vitro microscopy of beta cell lines has revealed the localization of molecular components in the cell and more recently their dynamic behavior. In cultured islets, interactions of beta cells with other islet cells and the matrix as well as paracrine and autocrine signaling or reaction to nutrients have been studied. Lastly, microscopy has been performed on tissue sections, visualizing the islets in an environment closer to their natural surroundings. In most efforts to date, the samples have been isolated for investigation and hence have by definition been divorced from their natural environments and deprived of vascularization and innervations. In such a setting the beta cells lack the metabolic information that is primordial to their basic function of maintaining glucose homeostasis. We review optical microscopy; its general principles, its impact in decoding beta cell function and its recent developments towards the more physiologically relevant assessment of beta cell function within the environment of the whole organism. This requires both large imaging depth and fast acquisition times. Only few methods can achieve an adequate compromise. We present extended focus Optical Coherence Microscopy (xfOCM) as a valuable alternative to both confocal microscopy and two photon microscopy (2PM), and discuss its potential in interpreting the mechanisms underlying glucose homeostasis and monitoring impaired islet function.

Bentham Science Publishers2010

Combined Optical Coherence and Fluorescence Microscopy to assess dynamics and specificity of pancreatic beta-cell tracers

Corinne Berclaz, Arno Pino Bouwens, Anne Grapin-Botton, Theo Lasser, Antonio Lopez, Christophe Pache, Daniel Pawel Szlag

The identification of a beta-cell tracer is a major quest in diabetes research. However, since MRI, PET and SPECT cannot resolve individual islets, optical techniques are required to assess the specificity of these tracers. We propose to combine Optical Coherence Microscopy (OCM) with fluorescence detection in a single optical platform to facilitate these initial screening steps from cell culture up to living rodents. OCM can image islets and vascularization without any labeling. Thereby, it alleviates the need of both genetically modified mice to detect islets and injection of external dye to reveal vascularization. We characterized Cy5.5-exendin-3, an agonist of glucagon-like peptide 1 receptor (GLP1R), for which other imaging modalities have been used and can serve as a reference. Cultured cells transfected with GLP1R and incubated with Cy5.5-exendin-3 show full tracer internalization. We determined that a dose of 1 μg of Cy5.5-exendin-3 is sufficient to optically detect in vivo the tracer in islets with a high specificity. In a next step, time-lapse OCM imaging was used to monitor the rapid and specific tracer accumulation in murine islets and its persistence over hours. This optical platform represents a versatile toolbox for selecting beta-cell specific markers for diabetes research and future clinical diagnosis.

2015

Definition of in Vitro Microenvironments to Characterize and Control Pancreatic Progenitor Expansion and Differentiation

Chiara Greggio

EPFL2011