To have a better understanding of the mechanisms involved during this in vitro islet generation we analyzed the gene expression profiles at different stages of the expansion/differentiation of these pancreatic cells: the Initial Adherent time point, the Expansion before changing to serum-free media and the final Differentiation stage at 4 weeks of culture (2 weeks after Matrigel) (Figure 1). The two time points that are the most interesting are the Expanded and Differentiated stages. As we reported previously, the Expanded stage is characterized by an almost confluent monolayer of epithelial cells that express CK19 and Pdx-1 with a minor component of small, triangular stromal cells. By the Differentiated stage, the epithelial cells have coalesced into thickened plaques and 3-dimensional structures of ductal cysts and budding islet-like structures. Between these structures, stromal cells have formed a near-confluent lawn. The tissue of a whole flask was taken for each sample, so the gene expression must be taken as of the whole system rather than of individual cells.

In contrast to LIST A, which is mainly comprised of genes encoding acinar-enriched enzymes, the genes in LIST B (Table 3) all have an increase in expression during expansion of the ductal epithelium (Expanded compared to the Initially Adherent stage). Genes regulating cell cycle progression such as CyclinD1, cyclin dependent kinases and cyclin dependent kinase inhibitors as well as numerous extracellular matrix genes (collagens and laminins), are present in this list. Genes of the TGFβ superfamily, such as TGFβ2, BMP and its antagonist gremlin, Smad 3 and 6, and members of Wnt family are also in this list. One genes of interest, DMBT-1 (deleted in malignant brain tumor 1), had greater than 5-fold increase in Expanded compared to the Initially Adherent stage. This gene encodes the protein for which a role in liver repair and recruitment of oval cells (liver stem cells) has been suggested (Bisgaard et al., 2002). We confirmed its expression in human pancreatic tissue by semi-quantitative RT-PCR (Figure 3) and by immunostaining. On frozen sections of human pancreas, DMBT-1 protein was only immunolocalized to scattered cells of the main duct and smaller peripheral ducts (Fig 4). In rat pancreas DMBT protein was only found in the epithelium of the small proliferating ductules of the newly forming lobes of regenerating rat pancreas (3 days after partial pancreatectomy) (Figure 5).

In contrast to LIST A, which is mainly comprised of genes encoding acinar-enriched enzymes, the genes in LIST B (Table 3) all have an increase in expression during expansion of the ductal epithelium (Expanded compared to the Initially Adherent stage). Genes regulating cell cycle progression such as CyclinD1, cyclin dependent kinases and cyclin dependent kinase inhibitors as well as numerous extracellular matrix genes (collagens and laminins), are present in this list. Genes of the TGFβ superfamily, such as TGFβ2, BMP and its antagonist gremlin, Smad 3 and 6, and members of Wnt family are also in this list. One genes of interest, DMBT-1 (deleted in malignant brain tumor 1), had greater than 5-fold increase in Expanded compared to the Initially Adherent stage. This gene encodes the protein for which a role in liver repair and recruitment of oval cells (liver stem cells) has been suggested (Bisgaard et al., 2002). We confirmed its expression in human pancreatic tissue by semi-quantitative RT-PCR (Figure 3) and by immunostaining. On frozen sections of human pancreas, DMBT-1 protein was only immunolocalized to scattered cells of the main duct and smaller peripheral ducts (Fig 4). In rat pancreas DMBT protein was only found in the epithelium of the small proliferating ductules of the newly forming lobes of regenerating rat pancreas (3 days after partial pancreatectomy) (Figure 5).

The final list (Table 5), List D, include those genes that are up-regulated during the Differentiated stage. As expected, the list includes genes encoding markers of terminally differentiated cells of the pancreas such as glucagon, insulin, IA2/phogrin and CFTR, mucin6 and 9 (ducts). Both glucagon and insulin were expressed at the Initially Adherent stage unlike the other members of the cluster, but their expressions decreased by Expanded stage before a more than 2-fold (re)induction by Differentiated stage. Interesting genes of this list include differentiation factors such as TGF-β superfamily member Gremlin, TGFβ1, BMP4 and 6, Wnt/frizzled genes Frizzled 1,2 and 7, Homeobox genes and the transcription factor c-maf known to play a role in differentiation β and α-cells. One gene of note PGP9.5/UHCL1, an ubiquitin carboxyl-terminal hydrolase, is a known neuronal marker and has been suggested to be a marker for islet precursor cells. PGP9.5 is expressed as early as E11.5 during development of the growing murine pancreas and later in adult islets (Yokoyama-Hayashi et al., 2002). Its expression was increased (more than 15-fold) from Expanded stage to the Differentiated stage. Semi-quantitative RT-PCR was performed using human PGP9.5 primers to confirm the array data (Figure 3). Similarly, immunohistochemical analysis was used to confirm the presence of PGP9.5 protein in both normal human and regenerating rat pancreas. Staining of cryostat sections of human pancreas revealed that islets and the epithelia of both small and large ducts were PGP9.5 positive (Figure 6). Additionally, the buds of endocrine tissue in the in vitro generated islet buds were positive (Figure 6C), and by double immunostaining, the expression of PGP9.5 was shown to be in the insulin positive cells (Figure 6D) rather than the glucagon positive cells (Figure 6E). In the rat regeneration model of 90 % partial pancreatectomy (Px + 3 days), PGP9.5 positive cells included most, if not all, islet cells, scattered cells in large ducts and in the pre-existing acini (possibly centroacinar cells) (Figure 7). In the newly forming lobes (Figure 7D, E), many of the epithelial cells in the small ductules were PGP9.5 positive.

During development many important pathways have been found to regulate proliferation and subsequent differentiation. During progression of the in vitro neogenesis as seen in this study, multiple members of two of these pathways, TGF-β Superfamily and the Wnt/Frizzled, were differentially expressed (Figure 8 and 9 respectively) suggesting that the pathways are important during in vitro islet formation from human pancreatic tissue. Selected genes in these pathways were confirmed with q-PCR: BMP1, BMP2A, FZD2, Gremlin and TGFB1. In addition FZD3, also a member of Wnt/frizzled pathway, which were not present on the genearray, showed a similar and interesting profile when using q-PCR (Figure 10).

In Figure 2, genes are organized into Scatter Plot (Figure 2A, top and bottom) and Heat Map (Figure 2B) according to expression level. In the Heatmap, the genes (across) are shown as normalized log scale values for each sample (down). In Scatter Plots, each dot represents the normalized average expression for one gene (The average expression of the four replicas, one from each pancreas). The diagonal lines represent the fold changes between the two stages, with the center diagonal line representing one fold change and the two outer diagonal lines either 2-fold increased or decreased. From the Scatter Plot four different scenarios were investigated: A: genes that lost expression from Initially Adherent to Expanded stage, B: genes with an increased expression with progression from the Initially Adherent though Expanded stage; C: genes that lose expression from the Expanded stage through the Differentiated stage; and D: genes that are more than 2-fold increased in Differentiated stage compared to Expanded stage. The following Tables do not list all of these genes but rather some of the more interesting ones with the p-value from t-test and fold change. The complete lists of differentially expressed genes with fold changes are given in the Supplementary data. The complete data sets have been deposited and can be accessed at http://www.cbil.upenn.edu/RAD/php/displayStudy.php?study_id=710&&download=1. As shown in Table 1, 1061 genes were more than 2 fold down regulated from Initially Adherent to Expanded stage. Similarly the table shows the overall genes present in the 3 other scenarios; lists of these genes are in Tables (2–5).

In contrast to LIST A, which is mainly comprised of genes encoding acinar-enriched enzymes, the genes in LIST B (Table 3) all have an increase in expression during expansion of the ductal epithelium (Expanded compared to the Initially Adherent stage). Genes regulating cell cycle progression such as CyclinD1, cyclin dependent kinases and cyclin dependent kinase inhibitors as well as numerous extracellular matrix genes (collagens and laminins), are present in this list. Genes of the TGFβ superfamily, such as TGFβ2, BMP and its antagonist gremlin, Smad 3 and 6, and members of Wnt family are also in this list. One genes of interest, DMBT-1 (deleted in malignant brain tumor 1), had greater than 5-fold increase in Expanded compared to the Initially Adherent stage. This gene encodes the protein for which a role in liver repair and recruitment of oval cells (liver stem cells) has been suggested (Bisgaard et al., 2002). We confirmed its expression in human pancreatic tissue by semi-quantitative RT-PCR (Figure 3) and by immunostaining. On frozen sections of human pancreas, DMBT-1 protein was only immunolocalized to scattered cells of the main duct and smaller peripheral ducts (Fig 4). In rat pancreas DMBT protein was only found in the epithelium of the small proliferating ductules of the newly forming lobes of regenerating rat pancreas (3 days after partial pancreatectomy) (Figure 5).

The final list (Table 5), List D, include those genes that are up-regulated during the Differentiated stage. As expected, the list includes genes encoding markers of terminally differentiated cells of the pancreas such as glucagon, insulin, IA2/phogrin and CFTR, mucin6 and 9 (ducts). Both glucagon and insulin were expressed at the Initially Adherent stage unlike the other members of the cluster, but their expressions decreased by Expanded stage before a more than 2-fold (re)induction by Differentiated stage. Interesting genes of this list include differentiation factors such as TGF-β superfamily member Gremlin, TGFβ1, BMP4 and 6, Wnt/frizzled genes Frizzled 1,2 and 7, Homeobox genes and the transcription factor c-maf known to play a role in differentiation β and α-cells. One gene of note PGP9.5/UHCL1, an ubiquitin carboxyl-terminal hydrolase, is a known neuronal marker and has been suggested to be a marker for islet precursor cells. PGP9.5 is expressed as early as E11.5 during development of the growing murine pancreas and later in adult islets (Yokoyama-Hayashi et al., 2002). Its expression was increased (more than 15-fold) from Expanded stage to the Differentiated stage. Semi-quantitative RT-PCR was performed using human PGP9.5 primers to confirm the array data (Figure 3). Similarly, immunohistochemical analysis was used to confirm the presence of PGP9.5 protein in both normal human and regenerating rat pancreas. Staining of cryostat sections of human pancreas revealed that islets and the epithelia of both small and large ducts were PGP9.5 positive (Figure 6). Additionally, the buds of endocrine tissue in the in vitro generated islet buds were positive (Figure 6C), and by double immunostaining, the expression of PGP9.5 was shown to be in the insulin positive cells (Figure 6D) rather than the glucagon positive cells (Figure 6E). In the rat regeneration model of 90 % partial pancreatectomy (Px + 3 days), PGP9.5 positive cells included most, if not all, islet cells, scattered cells in large ducts and in the pre-existing acini (possibly centroacinar cells) (Figure 7). In the newly forming lobes (Figure 7D, E), many of the epithelial cells in the small ductules were PGP9.5 positive.

During development many important pathways have been found to regulate proliferation and subsequent differentiation. During progression of the in vitro neogenesis as seen in this study, multiple members of two of these pathways, TGF-β Superfamily and the Wnt/Frizzled, were differentially expressed (Figure 8 and 9 respectively) suggesting that the pathways are important during in vitro islet formation from human pancreatic tissue. Selected genes in these pathways were confirmed with q-PCR: BMP1, BMP2A, FZD2, Gremlin and TGFB1. In addition FZD3, also a member of Wnt/frizzled pathway, which were not present on the genearray, showed a similar and interesting profile when using q-PCR (Figure 10).

During development many important pathways have been found to regulate proliferation and subsequent differentiation. During progression of the in vitro neogenesis as seen in this study, multiple members of two of these pathways, TGF-β Superfamily and the Wnt/Frizzled, were differentially expressed (Figure 8 and 9 respectively) suggesting that the pathways are important during in vitro islet formation from human pancreatic tissue. Selected genes in these pathways were confirmed with q-PCR: BMP1, BMP2A, FZD2, Gremlin and TGFB1. In addition FZD3, also a member of Wnt/frizzled pathway, which were not present on the genearray, showed a similar and interesting profile when using q-PCR (Figure 10).