Semb Laboratory: Human stem cell biology – University of Copenhagen

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The Semb Laboratory: Human stem cell biology

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Our lab has two main goals 1) to understand how cell polarity and tissue architecture control cell fate specification and 2) to translate this knowledge into efficient and reliable strategies for regenerative medicine in diabetes. These objectives are applied, primarily, to our organ of choice – the pancreas. We use a combination of mouse pancreatic epithelium and human stem cells as model systems to explore our goals, which, in turn, also serve as tools to understand pancreatic disease. Functioning as an interdisciplinary lab we work to combine knowledge from multiple systems; through the combinatorial use of animal models, stem cells and computer modelling we are exceptionally well placed to attain our goals.  Prof. H. Semb

 

The Semb lab has published pioneering results showing that apical polarity and normal pancreatic tissue architecture is required for beta cell specification (Kesavan et al.,2009). The emerging concept from this work is that apical polarity plays a central role in coordinating morphogenetic processes and cell fate specification. We believe that the challenge to generate mature glucose-responsive beta cells from human embryonic stem cells (hESCs) under current 2D culture conditions is due to a failure to regulate the epithelial intercellular signalling necessary for beta cell birth. We propose to establish a mechanism for how cell polarity within the multicellular pancreatic epithelium, both preceding and during lumen/tube formation, directly and indirectly leads to efficient beta cell birth in vivo. Further, we will translate these results into new 3D in vitro differentiation strategies of hESCs. These strategies will facilitate hESC-derived multi-potent pancreatic progenitors to undergo the necessary dynamic changes in cell polarity, cell adhesion and cell migration for functional beta cell birth.


On-going Developmental Biology Projects

The developing pancreas undergoes complex and intriguing epithelial cell rearrangements. The gross morphology of the organ changes from an epithelial sheet to an epithelial bud and finally to a branched/tubular epithelial tree. In parallel, pancreatic progenitors differentiate into multiple cell types and elicit major changes in cell organization including; microlumen and tube formation, acinus formation at the tips of the tubes and islet formation via delamination and clustering of new-born endocrine cells. It is becoming apparent that apical polarity links gross morphology, cellular rearrangement and cell differentiation as the pancreas forms. Our scientists are coordinating projects (see below) that address general developmental biology questions using pancreatic organogenesis as a model. Our primary focus is on the role of apical polarity.   

Novel tools for 3D live organ imagingWe have further developed an ex vivo system to study pancreatic morphogenesis and how it is linked to differentiation in pancreatic explants using confocal imaging. Taking advantage of existing cell membrane and stage-specific reporter mouse strains and combining them with our own newly generated reporter for apical polarity we can with single cell resolution monitor and quantify individual events. For instance, we are interested in tracking changes in cell polarity, microlumen formation over time and determining how these events affect fate decisions. We are aiming to implement this system to perform comparative live analysis of mouse models in which polarity is perturbed and to monitor the effects on tubulogenesis and cell differentiation.

In silico analysis of pancreatic cell behavioursIt is vital to quantify the cell traits we observe during our live image acquisitions. This is not a trivial task when we consider that the images are in 4D (3D plus time). To achieve this our computational biologist is writing new algorithms to segment cells and track migration. We strengthen our work in this field through our collaborations with additional experts at the computer science department of University of Copenhagen (DIKU). It is our aim to simulate and manipulate 4D cell behaviours in silico. Such computer models will enable us to ‘test’ many variables that could be attributed to specific cell behaviours, such as polarity expansion, before testing our hypotheses empirically.

Acquisition of apical polarity: The Semb lab has established that apical polarity is vitally important for both the formation of pancreatic tubes and the formation of endocrine cells. However, we still do not understand how apical proteins are recruited to the membrane of a pancreatic epithelial cell. Furthermore, we have yet to identify ‘apical membrane hierarchy’; by establishing which protein is first targeted to the epithelial membrane we could in turn identify unique regulators of this process. A number of projects in the lab are focusing on these issues with specific attention being paid to endocytotic pathways and the role of lipids within the epithelial membrane. Novel mouse and human pluripotent stem cell reporter lines (see below) are being established as tools to analyse these processes live.

Epithelial architecture and cell fate: Two fundamental questions within epithelial cell biology are, 1) how do epithelial cells exit from an epithelium and 2) how is cellular architecture (apical-basal polarity) and epithelial cell movement within the plane of an epithelium associated with a change in cellular phenotype? In a recent study our lab addressed question 1 and showed that pancreatic beta cell delamination and differentiation are independently controlled by Cdc42/N-WASP signalling (Kesavan et al., 2014). Building on this work and looking for new insights into delamination we have established projects that aim to understand the role of apical proteins and adherens junctions in this process. In addition to live imaging projects that address epithelial cell movement, we are also studying the link between apical polarity and beta cell differentiation. Our group has recently made an important observation that changing apical polarity autonomously affects the differentiation capacity of progenitors. We are currently extrapolating these in vivo results to hESCs in order to identify its impact on functional beta cell differentiation in vitro.

On-going Stem Cell Projects

As outlined above we are particularly interested in the potential of human pluripotent stem cells (hESCs and hiPSCs) as a source for mature insulin-producing beta cells for cell therapy in diabetes. The overall objective is to solve unanswered questions in endoderm and pancreas development biology and to use this knowledge to mass-produce transplantable insulin-producing beta cells for future cell therapy in diabetes. Although a number of signalling pathways are known to be important for pancreatic development, recapitulating these pathways in hESC differentiation is not sufficient to generate functional beta cells. Our lab is searching for novel links between signalling cascades that are necessary for full commitment of human pluripotent stem cells to the beta-cell lineage. We are also using hiPSCs to model monogenetic forms of diabetes.

Novel tools for in vitro stem cell analysis: We are currently using CRISPR/Cas9 as a tool to generate novel hESC reporter lines for apical membrane proteins and endocrine progenitors. These novel cell lines will allow us to visualise progenitor cells, their morphology and their polarisation status in parallel. This will enable us to analyse the connection between cell morphology and cell fate during hESC differentiation.

The importance of cell shape during differentiation: Changes in cell polarity and the architecture of an organ are linked to cell shape changes in vivo. Our scientists are exploring the requirement for distinct epithelial architecture for the birth of alpha and beta cells and whether certain niches have mechano-biological properties that favour specific endocrine fate decisions. We are performing screens (micro-pattern chips, hydrogels, phospho/proteomic screens and CRISPR/Cas9 KO screens) to identify conditions that will promote expansion or differentiation of multi-potent pancreatic progenitors by inducing changes in the shape of individual or multicellular 3D structures. In this project we also study the underlying molecular mechanism for how shape/3D cellular architecture and downstream signalling pathways influence cell behaviour via changes in intracellular tension. The long-term aim is to utilize this knowledge to facilitate generation of sufficient number of beta cells for clinical applications in cell therapy in diabetes.

Cell therapy in type-1 diabetes: The overall goal of the project is to manufacture GMP certified stem cell derived beta cells to initiate a phase 1 trial aiming to assess the safety of hPSC-derived beta cells. Based on recent discoveries in the Semb group we aim to address key technical hurdles, such as safety, purity and cost-effective scalability, that hinder stem cell therapy from becoming a clinically and commercially viable treatment in diabetes. The patent pending innovations are: 1) technologies allowing isolation of pancreatic beta cell progenitors from hPSCs (Ameri et al 2017), 2) new targets for controlling expansion of such progenitors (Ameri et al 2017), and 3) a novel approach to turn these progenitors into functional beta cells (Löf-Öhlin et al 2017, in revision; Mamidi, Prawiro et al, in preparation). Our immediate research will focus on GMP adapting our current differentiation protocol, establishing new strategies for efficient expansion of beta cell pancreatic progenitors, and improving beta cell maturation in vitro.  

iPSC disease modelling of monogenic forms of diabetes: Perturbation of pancreatic beta-cell function is generally regarded as the cause of type 2 diabetes (T2D), however, the exact mechanism behind these perturbations is not yet fully understood. In this project, we aim to establish in vitro models of monogenic hereditary forms of beta-cell dysfunction using iPSC lines from donors with Maturity Onset Diabetes of the Young (MODY). Isogenic control iPSC lines will be generated using CRISPR technology to correct the inherited mutations present in the MODY iPSC lines. By differentiating MODY iPSCs as well as the corrected isogenic controls into beta cells, we will establish a model system that can be used as a platform for drug screening and development of new treatments.

Lab Awards and Grants

PhD scientists:

Zarah Löf-Öhlin– SWEDBO (Swedish Developmental Biology Organization) conference, October 20th-21st, 2016, invited speaker.

Zarah Löf-Öhlin– BSCB/BSDB Joint Spring Meeting 2014 (University of Warwick) 16th-19th June; Invited speaker and ‘First prize in the BSDB Poster Prize’ competition.

Post-Doc Scientists:

Jacqueline Ameri (Assistant Professor) – Received in 2015 a Pre-Seed grant from Novo Nordisk Foundation for her project entitled “Developing methods for expandable
production of human pluripotent stem cell-derived insulin-producing beta cells for cell therapy in diabetes”. In 2016, she was also awarded a proof of concept grant from Copenhagen University to support the establishment of the spin-out company “DiaCure”, founded on IP generated by her research at DanStem.

Silja Heilmann – Was awarded “Det Frie Forskningsråd Sundhed og Sygdom” to develop segmentation and cell tracking algorithms for static and time-lapse 3D datasets of polarity in pancreata. The extracted information will be quantifiable and among other things, used to determine in silco model parameters. The subsequent Lundbeckfonden Postdoc in Denmark grant was awarded to develop mechano-chemical feedback functionality parameters in 3D pancreatic tissue models. These models will allow us to model cell phenotype/mechanical properties depending on local environmental constraints and on ‘memory’ of recent cellular deformations.

Anant Mamidi – The LundBeck Postdoctoral fellowship was granted to investigate a fundamental question in developmental biology, which is “what factors controls the organ size during development and injury?” To addresses this question the project primarily uses the mouse and human ESCs as model systems to analyse pancreatic progenitor maintenance and differentiation to endocrine lineage.

Pia Nyeng - 3 year postdoctoral fellowship from JDRF (Juvenile Diabetes Research Foundation) which is the leading global organization funding type 1 diabetes (T1D) research. JDRF Postdoctoral fellowships are designed to attract qualified, promising scientists entering their professional career in the diabetes research field.

The Semb Group

Selected Publications: