Semb Laboratory: Human stem cell biology – University of Copenhagen

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The Semb Laboratory: Human stem cell biology


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

The Semb lab has published ground-breaking 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 lack of progress in generating 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 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 multipotent 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 pancreas is an intriguing organ whose development is multi-dimensional. The gross morphology of the organ during embryogenesis changes from an epithelial sheet, to an epithelial bud and finally to a branched/tubular epithelial tree. In parallel, pancreatic progenitors must differentiate into multiple cell types and elicit major changes in cell organization including; microlumen formation (preceding the establishment of tubes), delamination, and islet formation. It is becoming apparent that apical polarity may link gross morphology, cellular rearrangement and cell differentiation as the pancreas forms. Our developmental biologists are coordinating projects (see below) that address each aspect of pancreas development, with a primary focus on the role of apical polarity.   

Novel tools for ex vivo analysis: 3D live organ imaging: We have further developed an ex vivo system to study pancreatic morphogenesis and how it is linked to differentiation in whole 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, such as changes in cell polarity, microlumen formation and the link between these events and fate decisions over time. 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.

Acquisition of apical polarity: The Semb lab has established 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 an ‘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 reporter lines 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 recently accepted paper in the journal Development, our lab has addressed question 1 and shown 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, affects the differentiation capacity of progenitors. We are currently extrapolating these in vivo results to hESC in order to identify its impact on functional beta cell differentiation in vitro. In addition, our scientists are exploring the requirement for distinct epithelial architecture for the birth of Ngn3+ cells and whether certain niches have mechano-biological properties that favour endocrine differentiation.

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 to identify, novel links between signalling cascades that may be 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 have generated pancreas specific reporter cell lines (eg PDX1-GFP and NGN3-GFP) using classical gene targeting by homologous recombination. These reporter lines are useful tools to study  the molecular mechanisms for expansion of multipotent human pancreatic progenitors and their differentiation into glucose-responsive beta cells via Ngn3 endocrine progenitors. These resources are also used to identify new cell surface markers for distinct beta cell progenitors.

The importance of cell shape during stem cell differentiation: Changes in cell polarity and the architecture of an organ are linked to cell shape changes in vivo. It has been shown that shape influences cellular decisions  such as proliferation and differentiation, of many tissue-specific stem cells. We are performing screens to identify conditions that will promote expansion or differentiation of multipotent 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 architecture influence cell behaviour via changes in intracellular tension. The long-term aim to utilize this knowledge to facilitate generation of sufficient number of beta cells for clinical applications in cell therapy in diabetes.

WNT signalling in human germ layer specification: WNT signalling plays a pivotal role in germ layer specification in the early mouse embryo, yet little is known about the underlying mechanism and whether this requirement is conserved for human development. Our group has used hESCs as a model to address these questions and has found that canonical WNT signalling collaborate with other self-renewal and differentiation pathways to regulate primitive streak and definitive endoderm fate decisions. The practical application of these findings includes improved efficiency of generating definitive endoderm and pancreatic lineages from hESCs/hiPSCs.

Isolation and molecular characterization of multipotent human pancreatic beta cell progenitors: We conduct gene expression analysis of isolated pancreatic progenitors (single cell and population-based analysis) to define novel cell surface markers. After validation of these markers the lineage potential of the isolated cell populations are studied and utilized to develop more defined differentiation protocols of hESCs into beta cells.  

iPS disease modeling 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 3 (MODY3). Isogenic control iPSC lines will be generated using CRISPR technology to correct the inherited mutations present in the MODY3 iPSC lines. By differentiating MODY3 iPS cells as well as the corrected isogenic controls into beta cells, we hope to establish a model system that can be used as a platform for drug screening and to develop new treatments for this form of T2D.

Lab Awards and Grants

PhD scientists:
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:
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 my project primarily uses the mouse as a model system to analyse pancreas morphogenesis and cell specification.
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.
Oliver Leiven – Beta Cell Biology Consortium (BCBC) investigator retreat 7th-9th May 2013, Washington; Invited speaker.

Selected Publications

Kesavan, Gokul, Oliver Lieven, Anant Mamidi, Zarah Lof Ohlin, Jenny Kristina Johansson, Wan-Chun Li, Silvia Lommel, Thomas Uwe Greiner & Henrik Semb (2014). Cdc42/N-WASP signaling links actin dynamics to pancreatic beta cell delamination and differentiation. Development, 141(3), 685-696, doi:10.1242/dev.100297. 
We identified for the first time a direct link between junctional actin dynamics via Cdc42/N-WASP signaling and transcriptional regulation of pancreatic beta cell differentiation.”

Hernebring, Malin, Åsa Fredriksson, Maria Liljevald, Marija Cvijovic, Karin Norrman, John Wiseman, Henrik Semb & Thomas Nyström (2013). Removal of damaged proteins during ES cell fate specification requires the proteasome activator PA28. Scientific Reports, 3, 1381, doi: 10.1038/srep01381. 
“In this paper we provide a mechanism for how damaged proteins are removed during ES cell differentiation.”         

Sand, Fredrik Wolfhagen, Andreas Hörnblad, Jenny K. Johansson, Christina Lorén, Josefina Edsbagge, Anders Ståhlberg, Judith Magenheim, Ohad Ilovich, Eyal Mishani, Yuval Dor, Ulf Ahlgren & Henrik Semb (2011). Growth-limiting role of endothelial cells in endoderm development. Developmental Biology 352(2), 267-277, doi:10.1016/j.ydbio.2011.01.026. 
We showed that endothelial cells limit growth of foregut-derived organs, including the pancreas.”

Fischer, Yvonne, Elvira Ganic, Jacqueline Ameri, XiaoJie Xian, Martina Johannesson & Henrik Semb (2010). NANOG Reporter Cell Lines Generated by Gene Targeting in Human Embryonic Stem Cells. Plos One, 5(9), e12533, doi:10.1371/journal.pone.0012533. 
“We report generation of NANOG reporter hESC lines using gene targeting by homologous recombination to elucidate the regulation and function of NANOG in pluripotent hESCs.”

Ameri, Jacqueline, Anders Ståhlberg, Jesper Pedersen, Jenny K. Johansson, Martina M. Johannesson, Isabella Artner & Henrik Semb (2010). FGF2 Specifies hESC-Derived Definitive Endoderm into Foregut/Midgut Cell Lineages in a Concentration-Dependent Manner. Stem Cells, 28(1), 45-56, doi:10.1002/stem.249. 
“We showed for the first time that FGF2 specifies hESC-derived definitive endoderm into different foregut lineages (liver, pancreas, lung) in a dosage-dependent manner.”

Kesavan, Gokul; Fredrik Wolfhagen Sand, Thomas Uwe Greiner, Jenny Kristina Johansson, Sune Kobberup, Xunwei Wu, Cord Brakebusch & Tor Henrik Semb (2009). Cdc42-mediated tubulogenesis controls cell specification. Cell, 139(4), 791-801, doi:10.1016/j.cell.2009.08.049. (Highlighted by the “Faculty of 1000”).
“We showed that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity in vivo, and that lumens/tubes provide the correct microenvironment for proper control of cell-fate choices of multipotent progenitors.”

Ellerström, Catharina, Raimund Strehl, Karina Moya, Katarina Andersson, Christina Bergh, Kersti Lundin, Johan Hyllner & Henrik Semb (2006). Derivation of a xeno-free human embryonic stem cell line. Stem Cells, 24(10), 2170-2176, doi:10.1634/stemcells.2006-0130. 
“We report the establishment and characterization of a xeno-free pluripotent diploid normal hESC line.”

Xian, Xiaojie, Joakim Håkansson, Anders Ståhlberg, Per Lindblom, Christer Betsholtz, Holger Gerhardt & Henrik Semb (2006). Pericytes limit tumor cell metastasis. Journal of Clinical Investigation, 116(3), 642–651, doi:10.1172/JCI25705. 
“We discovered a new model for how tumor cells trigger metastasis by perturbing pericyte-endothelial cell-cell interactions.”

Brolén, Gabriella K.C., Nico Heins, Josefina Edsbagge & Henrik Semb (2005). Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells. Diabetes, 54(10), 2867-2874, doi:10.2337/diabetes.54.10.2867. 
One of the first studies showing the capacity of hESCs to differentiate into insulin-producing beta cells in response to cues from the developing mouse pancreas.”

Edsbagge, Josefina, Jenny K. Johansson, Farzad Esni, Yang Luo, Glenn L. Radice & Henrik Semb (2005). Vascular function and sphingosine-1-phosphate regulate development of the dorsal pancreatic mesenchyme. Development, 132(5), 1085-92, doi: 10.1242/dev.01643. 
“Our data support a new model for how blood vessel-derived sphingosine-1-phosphate stimulates growth and budding of foregut-derived endodermal organs, such as the pancreas, by induction of mesenchymal cell proliferation.”

Perl, Anne-Karina, Ulf Dahl, Petra Wilgenbus, Harold Cremer, Henrik Semb & Gerhard Christofori (1999). Reduced expression of neural cell adhesion molecule induces metastatic dissemination of pancreatic beta tumor cells. Nature Medicine, 5, 286-291, doi:10.1038/6502. 
We showed that the loss of NCAM-mediated cell adhesion is one rate-limiting step in the actual metastatic dissemination of beta tumor cells.”

Esni, Farzad, Inge-Bert Täljedal, Anne-Karina Perl, Harold Cremer, Gerhard Christofori & Henrik Semb (1999). Neural cell adhesion molecule (N-CAM) is required for cell type segregation and normal ultrastructure in pancreatic islets. Journal of Cell Biology, 144(2), 325-337 doi: 10.1083/jcb.144.2.325. 
“We confirmed for the first time in vivo that cell adhesion molecules, in this case N-CAM, is required for cell type segregation during.”

Perl, Anne-Karina, Petra Wilgenbus, Ulf Dahl, Henrik Semb & Gerhard Christofori (1998). A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature, 392(6672), 190-193, doi:10.1038/32433. 
“Our results demonstrated that loss of E-cadherin-mediated cell adhesion is one rate-limiting step in the progression from adenoma to carcinoma.”