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Keywords:

  • embryonic stem cells;
  • endoderm;
  • in vitro differentiation;
  • induced pluripotent stem cells;
  • pancreas

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES

Embryonic stem (ES) cells or induced pluripotent stem (iPS) cells are expected as a surrogate cell source for regenerative medicine. Many researchers have reported the differentiation method of insulin-expressing pancreatic β cells from ES or iPS cells. However, the detailed molecular mechanisms underlying the differentiation of ES or iPS cells into pancreatic lineages are still unclear. We have established a feeder cell-based differentiation system into pancreatic progenitor cells, and revealed the signaling pathways that are involved in the differentiation of ES cells into mesendoderm, endoderm and pancreatic progenitor cells. Recently, we demonstrated that the extracellular environment, particularly the laminin-integrin signaling and heparan sulfate proteoglycan, is important for the regionalization of definitive endoderm cells into pancreatic lineages. These results provide new insights for the differentiation mechanism of pancreatic cell lineages.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES

Embryonic stem (ES) cells have an unlimited replicative ability and the potential to differentiate into most cell types in an organism, including pancreatic cell lineages. Recently, it was reported that the introduction of several transcription factors induce the reprogramming of somatic cells into induced pluripotent stem (iPS) cells (Takahashi et al. 2007; Nakagawa et al. 2008). These materials are expected not only as a source of regenerative medicine, but also as an in vitro differentiation model of early developmental steps. As organ-specific progenitor or precursor cells are limited in numbers in the embryo, ES cell-derived organ-specific progenitor or precursor cells would be a powerful tool for analyzing the developmental characteristics of progenitor cells.

Many researchers have reported the differentiation procedures for pancreatic cell lineages from ES or iPS cells. It has been demonstrated that ES or iPS cells are sequentially differentiated into pancreatic cells following normal developmental processes. ES or iPS cells first differentiate into mesendoderm, a common bipotent progenitor of the definitive endoderm and mesoderm. Mesendoderm then diverge into a definitive endoderm or mesoderm fate. Then, pancreatic progenitor cells arise from the definitive endoderm. Several groups have reported in vitro differentiation of mouse ES cells into mesendoderm (Tada et al. 2005), definitive endoderm (Kubo et al. 2004; Yasunaga et al. 2005; Gadue et al. 2006) or foregut endoderm (Hansson et al. 2009). Other groups have claimed the generation of definitive endoderm (D'Amour et al. 2005), pancreatic progenitors (Johannesson et al. 2009) or insulin-expressing cells (D'Amour et al. 2006; Jiang et al. 2007) from human ES cells. In these studies, human ES cell-derived pancreatic cells showed insulin secretion in response to various reagents, but not to glucose in vitro (D'Amour et al. 2006). However, Kroon et al. showed that human ES cell-derived pancreatic progenitor cells further matured into functional β cells, which responded to glucose stimulation in vivo (Kroon et al. 2008). These results indicate that in vivo differentiation is anticipated to provide a suitable environment for further maturation of ES cell-derived pancreatic cells. Recently, large-scale screenings of small molecules have identified chemical compounds that promote ES cell differentiation into definitive endoderm (Borowiak et al. 2009) or pancreatic progenitor cells (Chen et al. 2009). Such small molecules are useful for their potential applications for regenerative medicine in in vitro differentiation studies, or the identification of molecular mechanisms that regulate gut regionalization into the pancreas or further differentiation of pancreatic progenitor cells into mature pancreatic endocrine or exocrine cells that express insulin, glucagon or amylase.

Although the abovementioned reports showed that definitive endoderm cells and insulin-producing cells could be generated in vitro from mouse and human ES cells, the molecular mechanisms for the inductive process remain largely unknown. To reveal the developmental process of pancreatic cells, we established in vitro differentiation systems into pancreatic cell lineages. We demonstrated that several specific signaling molecules (activin, bFGF, retinoic acid) and an extracellular environment were important for the differentiation of pancreatic progenitor cells.

A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES

The pancreas consists of endocrine, exocrine and ductal cells. All of these cell lineages are derived from pancreatic progenitor cells that express Pancreatic duodenum homeodomain 1 (Pdx1). Pdx1 is a regional endoderm marker, which marks the dorsal and ventral pancreatic buds, as well as part of the stomach and duodenal endoderm (Offield et al. 1996). Signals from adjacent germ layers are important for the differentiation of Pdx1-expressing pancreatic progenitor cells (Wells and Melton 1999; Lammert et al. 2001). We speculated that a culture cell line derived from mesoderm could induce the differentiation of ES cells into pancreatic lineages. We examined several cell lines and found that M15, a mesonephros-derived cell line, served as an excellent endoderm inductive source (Shiraki et al. 2008).

On culturing mouse ES cells on a monolayer of M15 cells, ES cells are differentiated into Pdx1-expressing pancreatic progenitor cells (Fig. 1A). The differentiation of ES cells into Pdx1-expressing cells is a multistep process. ES cells are diverged into a mesendoderm or ectoderm fate (early phase). Then, the bipotential mesendoderm are diverged into a mesoderm or definitive endoderm fate (middle phase). The definitive endoderm is then turned into regional-specific tissue of the endoderm (late phase). On day 8 of differentiation on M15, Pdx1-expressing cells are observed to give rise in the edge of the colonies. The molecular bases of the signaling event involved in each step of the processes are summarized in Figure 1B. The efficient generation of definitive endoderm can be achieved by adding basic FGF (bFGF) and activin, which sequentially inhibit differentiation of ES cells into ectoderm and mesoderm. Conversely, inhibiting these pathways can specifically trigger neuroectodermal or mesodermal lineage differentiation. The M15 procedure, therefore, provides a potentially useful tool enabling investigations into the molecular mechanisms regulating the divergence of ES cells into different germ layers. We performed microarray analysis of ES cell-derived ectoderm, mesendoderm, mesoderm, and definitive endoderm cells at various time points. We revealed that ES cell-derived germ layer cells express germ layer-specific marker genes, in a manner that mimics early normal embryonic development (Shiraki et al. 2009). Moreover, we discovered decay accelerating factor (DAF1/CD55) as a candidate surface marker for the identification of the early and late definitive endoderm (Shiraki et al. 2010). These results strongly suggest that the M15 differentiation system is useful to analyze early developmental steps in vitro.

image

Figure 1. M15-based differentiation procedure. (A) Schema of differentiation. Mouse embryonic stem (ES) or induced pluripotent stem (iPS) cells are seeded on Mitomycin C-treated M15 cells, and cultured with activin A and bFGF-containing medium until differentiation day 8 (d8), on which green fluorescence represents the expression of Pdx1. Bar, 200 µm. (B) Schematic drawing of the signaling molecules involved in the differentiation of ES cells into three germ layers and pancreatic progenitor.

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Activin and bFGF, which are known as notochordal signals, maintain Pdx1 expression in the endoderm, and in turn potentiate pancreatic differentiation (Wells and Melton 2000). On M15, retinoic acid acts as an ectoderm inducer in the early phase, but as a pancreatic inducer at the late phase (Shiraki et al. 2008). As activin and bFGF promote ES cell differentiation at all phases of induction, activin and/or bFGF added throughout the entire processes of ES differentiation in our M15 systems, namely days 0–8, resulted in a maximum yield of Pdx1-expressing cells (∼30% of differentiated cells express Pdx1). The differentiation potential of the ES cell-derived pancreatic progenitor cells was tested by engraftment under the kidney capsule of severe combined immunodeficiency (SCID) mice (Shiraki et al. 2008). Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemical analysis of the grafts revealed that the endocrine lineage markers, such as Neurogenin3, Insulin1, Glucagon, and Pancreatic polypeptide, as well as the exocrine marker Amylase, duct marker Cytokeratin 19, along with expression of other β cell markers, such as Islet amyloid polypeptid, Kir6.2, and Glut2, increased after transplantation. Immunohistochemical analysis revealed that C-peptide was detected in the Pdx1-positive cell after transplantation. Taken together, ES cell-derived pancreatic progenitor cells obtained under our present in vitro differentiation system have the potential to differentiate into all pancreatic lineages, namely endocrine, exocrine, and duct cells. However, after transplantation of the differentiated ES cell-derived Pdx1-expressing cells into streptozotocin-induced diabetic mice, reversal of blood glucose was not observed (unpubl. data). Therefore, further improvement of the pancreatic maturation step either in vitro or in vivo is required for the cells to acquire the ability to secrete insulin in a glucose-dependent manner.

IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES

From the M15 study, we found that the late phase, namely the regional differentiation into cells of various digestive organs, required a close contact of the ES cells with M15 (Shiraki et al. 2008). This was shown by a trans-filter assay, in which ES cells and M15 were cultured separately in the upper or lower chambers. ES cells differentiated into definitive endoderm, but not further into Pdx1-expressing cells. Moreover, fixed M15 cells retained the inducing ability of pancreatic progenitor cells. These results demonstrated that cell-cell interaction is important for pancreatic differentiation.

Microarray analysis of M15 cells revealed high expression levels of collagen type IV and laminin α5 (Lama5), which are major components of basement membrane. M15 cells also highly express type V collagen of lamina fibroreticulas. Basement membrane (BM) has a highly integrated structure composed of extracellular matrix (ECM) molecules, and plays important roles in various kinds of cellular functions, such as adhesion, migration, proliferation and cell differentiation (Yurchenco and Schittny 1990; Taipale and Keski-Oja 1997). The major components of most basement membranes are type IV collagen, laminins (LNs), entactin (nidogen) and heparan sulfate proteoglycans (Hspg), such as perlecan. Among these components, LNs serve as the major adhesive proteins and mediate cell adhesion to BM (Kleinman et al. 2003). These molecules are assembled to form an optimal extracellular environment for developing, regenerating or maturing cells. To test whether the BM produced by M15 plays an important role in the regional specification of definitive endoderm cells, we tested the effect of inhibition of Lama5 expression in M15. As a result, the potential of M15 to support the differentiation of ES cells into Pdx1-expressing cells was significantly reduced when Lama5 expression was reduced (Higuchi et al. 2010). These results suggest the importance of BM components in the process of pancreatic differentiation and the potential of using ECM as an inducer of pancreatic differentiation.

Previously, Mochitate and colleagues investigated the basement membrane formation by culturing epithelial cells in vitro (Furuyama and Mochitate 2000) and reported the terminal differentiation of tracheal basal cells into ciliated cells on a novel substratum, synthesized basement membrane (sBM) (Hosokawa et al. 2007). We applied this substratum for the pancreatic differentiation of ES cells. As Lama5 mediates the guiding signal from M15, we reconstituted the ECM environment using sBM prepared from rLN-10 cells, which constitutively express LN511 (consists of laminin α5, β1 and γ1). As expected, mouse ES or iPS cells were sequentially induced into definitive endoderm, pancreatic progenitor and insulin-expressing cells on rLN-10 sBM (Fig. 2A). When transplanted under kidney capsules of SCID mice, the ES cells further differentiated into mature pancreatic cell lineages, and formed islet-like clusters in vivo (Higuchi et al. 2010).

image

Figure 2. Synthesized basement membrane (sBM)-based differentiation procedure. (A) Schema of the differentiation procedure. Mouse embryonic stem (ES) or induced pluripotent stem (iPS) cells are seeded on sBM and cultured. Expression of pancreatic duodenum homeodomain 1 (Pdx1) is observed on day 15 (d15, indicated as green fluorescence). Expression of Insulin1 (Ins1) is observed at day 28 (d28), (indicated as green fluorescence). Bars, 100 µm. (B) Differentiation signals exerted from the basement membrane. ES cells receive extracellular signals through interaction of integrin β1 (Itgb1) with laminin α5 (Lama5). Together with heparan sulfate proteoglycans 2 (Hspg2), which is secreted from the ES cells and integrated into the basement membrane. The basement membrane components mediate signals that potentiate pancreatic differentiation of ES cells. Moreover, various kinds of signaling molecules are trapped in the heparan sulfate chain of Hspg2 or other Hspgs. These molecules directly and/or indirectly induced pancreatic differentiation.

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We then focused on the molecular mechanism of how basement membrane directs the pancreatic differentiation. As knockdown of Lama5 in M15 led to a reduction in the number of the Pdx1-expressing pancreatic progenitor cells, we speculated that Lama5-containing LNs mediated the guiding signal from the extracellular environment. We then performed a knockdown assay of LN receptor, integrin β1 (Itgb1) in the ES cells. As a result, down-regulation of Itgb1 inhibited the differentiation of pancreatic progenitor cells. These results suggest that the guiding signal from sBM was transduced through Itgb1. It is reported that pancreatic β cells express Itgb1, and vascular endothelial cells in the islet provided BM containing LN411 and LN511 (Nikolova et al. 2006). In the adult islet, the interaction of LNs with integrin regulates insulin synthesis. Taken together, these findings indicate that LN-integrin signaling affects not only the adult β cell function, but also early pancreatic differentiation.

As down-regulation of LN-integrin signaling partially decreased the number of Pdx1-expressing cells, we speculated that other molecules might also be involved in this process. We then focused on Hspgs. It is known that heparan sulfate chains of Hspgs act as a reservoir or modulator for various kinds of growth factors and signaling molecules (Izvolsky et al. 2003; Wu et al. 2004). Hspgs are divided into two groups: those that exist in the cell surface (such as syndecan and glypican) and those that exist in the basement membrane. In particular, Hspg2 is known to be the main BM component. Hspg2 knockdown and degradation of heparan sulfate chains did not affect the differentiation of ES cells into the definitive endoderm, but synergistically decreased the differentiation into Pdx1-expressing cells. FGFs, FGF receptors and Hspgs are reported to form a ternary complex, which confers the ligand-receptor specificity of FGF signaling (Rodgers et al. 2008). Moreover, selective binding of VEGFs to Hspgs is shown to control the growth and shaping of the vascular trees (Ruhrberg 2003). FGFs and VEGFs are pancreatic inducing signals secreted from the notochord and β cell inducing signals secreted from the dorsal aorta, respectively (Wells and Melton 1999; Lammert et al. 2001; Yoshitomi and Zaret 2004). Therefore, our results strongly indicate that the heparan sulfate chain might participate in mediating signaling transduction through growth factors, such as FGF and VEGF. Taken together, these findings suggest that Hspg2 and other Hspgs regulate the differentiation of pancreatic cell lineages.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES

We have established a differentiation procedure of pancreatic progenitor cells using M15 cells as an inducer. We revealed the molecular mechanism of inductive signals from M15, and found that activin, FGF and RA signaling particularly enhanced the differentiation of ES cells into mesendoderm, definitive endoderm and pancreatic progenitor cells. Furthermore, we established a novel feeder-free differentiation procedure using sBM and demonstrated that an extracellular environment is important for the regional specification of the definitive endoderm cells into pancreatic progenitor cells. We revealed that the differentiation signals from BM are transduced in part through LN-integrin, and in part through growth factor receptors, which function in the presence of Hspg2 or other Hspgs in BM (Fig. 2B). These findings provide us new insights of early pancreatic differentiation process. These procedures will be a useful tool not only for the elucidation of the molecular mechanisms, but also as an attractive approach for generating surrogate cell sources for applications in regenerative medicine.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. A MESONEPHRIC CELL LINE, M15, CAN INDUCE MOUSE ES OR IPS CELLS INTO PANCREATIC CELL LINEAGES
  5. IMPORTANCE OF EXTRACELLULAR ENVIRONMENT FOR PANCREATIC DEVELOPMENT
  6. CONCLUSION
  7. REFERENCES