Materials and methods
The roles of endogenous stem/progenitor Flavopiridol hydrochloride in adult pancreatic tissue homeostasis, acute/chronic injury response and tumor initiation remain uncertain. However, it is now clear that a subset of pancreatic duct cells are capable of initiating long-term self-renewing organoid cultures with effectively unlimited potential for expansion in culture. The M+133+26− subsets of both the pancreas and liver of adult mice contain the only cells with this capability. Lgr5, which is a marker for self-renewing stem cells in the colon, small intestine and stomach only becomes expressed after injury in the adult liver and pancreas (Barker et al., 2007, 2010; Huch et al., 2013a,b). Hence, in these latter tissues Lgr5 is only a marker for activated progenitors and cannot be used to prospectively identify clonogenic cells in normal, uninjured tissue. Our study is the first to report the prospective purification of “inactive” pancreatic and liver mouse organoid forming progenitors. We applied population expression profiling with RNA-seq and single cell analyses to determine the transcriptomes and transcriptional heterogeneity of these important populations. The ability to purify both inactive and injury-activated progenitors will permit the study of the molecular circuitry underlying progenitor activation in the future.
Although heterogeneity of pancreatic ducts has long been known based on morphological criteria, we demonstrate here for the first time that pancreatic duct cells are heterogeneous in terms of clonogenic potential. Thus it is appropriate to distinguish between clonogenic and non-clonogenic ducts. The surface marker combinations used herein do not permit the unambiguous identification of these duct subsets in histological sections. Hence, we currently do not know whether clonogenic and non-clonogenic ducts reside in anatomically distinct locations or whether they are intermingled and lie adjacent to each other. Our RNA-seq analysis represents a list of distinctive markers that could be used to localize the progenitors in tissue sections and generate lineage-tracing tools in the future. The observation of high Sox9 expression in only a small subset of M+133+26− is interesting, and it is tempting to speculate that this property is predictive of their organoid-forming capacity. We note that heterogeneity of Sox9 at the protein level has recently been reported in epithelial tissues. Ramalingam et al. found that intestinal epithelial cells express Sox9 at distinct levels, and that only the highest expressing subpopulation had progenitor activity (Ramalingam et al., 2012).
We have recently shown that gall bladder derived epithelial cells can be reprogrammed to become endocrine-like and express insulin using a combination of Pdx1, Ngn3 and MafA (Hickey et al., 2013). Others have shown that biliary duct cells in the liver can be reprogrammed in vivo and cure diabetes in mice (Akinci et al., 2012). Here we demonstrate that pancreatic duct cells can similarly be reprogrammed even after massive expansion in organoid culture. The expandability of the pancreatic progenitors makes them a potential target for generating transplantable beta cells. Ducts and acinar cells are routine by-products of islet isolation procedures using in clinical transplantation (Baeyens and Bouwens, 2008). Recently, the laboratory of S. Kim has shown that human ducts can also be reprogrammed to become functional beta-like cells (Lee et al., 2013). Therefore, the progenitor population described here may have potential utility in beta cell replacement therapy for diabetes.
The following are the supplementary data related to this article.
Primary cilia (non-motile) are cellular extensions extending from most growth arrested mammalian cells and are responsible for coordinating responses to extracellular conditions (Basten and Giles, 2013; Christensen et al., 2008; Quarmby and Parker, 2005; Satir et al., 2010). Primary cilia sense the extra-cellular environment by chemosensation and mechanotransduction, transducing this information through signaling cascades, allowing cells to respond to environmental stimuli (Avasthi and Marshall, 2012; Davenport and Yoder, 2005; Han et al., 2008; Malone et al., 2007; Oh and Katsanis, 2012). The primary cilium is comprised of two main parts: 1) a basal body, consisting of modified centrioles and 2) a ciliary axoneme, comprised of a ring of 9 microtubule doublets bound by the cell membrane. As sensory organs primary cilia [9(+0)] lack the central structural microtubule doublet (+2) and dynein arms required for motile cilia [9(+2)] (Basten and Giles, 2013; Takeda and Narita, 2012). Importantly, defects in primary cilia have been found to be the causative agent behind many human diseases or developmental defects (Nigg and Raff, 2009; Oh and Katsanis, 2012; Pan et al., 2005; Schmidt et al., 2012; van Reeuwijk et al., 2011; Weatherbee et al., 2009; Zalli et al., 2012).
Materials and methods