Abstract
β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed “cobblestone” morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed “fibroblast-like” morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6–10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.
Keywords
Introduction
Patients with type 1 diabetes and some cases of type 2 diabetes experience a reduction of insulin secretion that is linked to a significant decrease in the number of pancreatic β-cells. Therefore, it is important to search for ways to obtain sufficient insulin-producing cells for transplantation in diabetes. Islet transplantation is a promising possibility for the optimal treatment of type 1 diabetes (9, 10, 20, 23, 29). However, an abundant source of tissue that satisfies the demand for β-cells has yet to be found. One attractive approach for the generation of β-cells involves the expansion and differentiation of adult human pancreatic stem/progenitor cells, which are closely related to the β-cell lineage. Several studies have suggested that adult β-cells might originate from ductal or duct-associated cells (1, 3, 4, 13, 14, 32).
It has recently been reported that differentiated ductal cells, which express carbonic anhydrase II, could act as progenitors that give rise to both new islets and acini (normally after birth and after injury) (6). Ductal progenitor cells in the pancreas would become a particularly useful target for therapies that target β-cell replacement in diabetic patients (29, 30) because ductal cell types are abundantly available in the pancreata of these patients and in donor organs. However, their limited survival in culture and their low ability to differentiate into insulin producing cells in vitro are major issues.
We recently established a mouse pancreatic stem cell line without genetic manipulation (19). The clonal cell line obtained, HN#13, expresses the pancreatic and duodenal homeobox factor-1 (PDX-1), also known as IDX-1/STF-1/IPF1, one of the transcription factors of β-cell lineage. Induction therapy with exendin-4 and with PDX-1 and BETA2/NeuroD transcription factors using protein transduction technology (13, 14, 17, 18, 31) stimulated the expression of insulin mRNA in the cells. Yamamoto et al. also reported mouse pancreatic stem cells using serum-free medium containing cholera toxin, which stimulates cAMP signaling in cells (32). The pancreatic stem cells have the potential to differentiate into not only insulin-producing cells but also hepatocytes (19, 32). These isolation and culture techniques might be useful for identification and isolation of human pancreatic stem/progenitor cells.
In this study, we used these techniques to identify and isolate human pancreatic stem/progenitor cells. We also tested whether exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into human pancreatic stem/progenitor cells induced insulin and pancreas-related gene expression.
Materials and Methods
Human Islet Isolation
The islet isolation protocol was approved by the Institutional Review Board of Baylor Health Care System. All pancreata were procured using a standardized technique to minimize warm ischemia. University of Wisconsin (UW) solution was used for in situ perfusion of the donor. The pancreas was excised immediately after the liver and before the kidneys and was placed on ice. After the removal of the spleen and duodenum, we immediately inserted a cannula into the main pancreatic duct. The pancreas was weighed and 1 ml/g pancreas weight of modified Kyoto solution (Kyoto solution with ulinastatin; Kyoto solution, Otsuka Pharmaceutical Factory, Inc., Naruto, Japan; ulinastatin, Mochida Pharmaceutical, Tokyo, Japan) was infused through an intraductal cannula (9, 22). Pancreata were placed into a modified Kyoto solution/perfluorochemical two-layer preservation container at 4°C (15, 20, 21) for less than 8 h until the islet isolation procedure.
Human islet isolation was conducted as previously described in the standard Ricordi technique (9, 10, 16, 28) with modifications introduced in the Edmonton protocol (11, 27, 29). In brief, after decontamination of the pancreas, the ducts were perfused in a controlled fashion with a cold enzyme blend of Serva collagenase NB1 containing neutral protease (SERVA Electrophoresis GmbH, Heidelberg, Germany). The distended pancreas was then cut into 7–9 pieces, placed in a Ricordi chamber, and shaken gently. While the pancreas was being digested by recirculating the enzyme solution through the Ricordi chamber at 37°C, we monitored the extent of digestion with dithizone staining by taking small samples from the system. Once digestion was confirmed to be complete, dilution solution (Mediatech, Inc., Manassas, VA) was introduced into the system. Then, the system was cooled to stop further digestive activity. The digested tissue was collected in flasks containing 25% HSA and washed with fresh medium to remove the enzyme. Islets were purified with a continuous density gradient of iodixanol-based solution (Optiprep®, Sigma-Aldrich, St. Louis, MO) in an apheresis system (COBE 2991 Cell Processor, Gambro Laboratories, Denver, CO) as previously reported (9, 16).
Isolated islets from human pancreata were cultured at 37°C and 5% CO2 in culture medium. Culture medium was CMRL-based Miami-defined media #1 (MM1; Mediatech-Cellgro, Herndon, VA) containing 0.5% human serum albumin, which has been used for human islet transplantation previously (5).
Culture of Human Pancreatic Progenitor Cells
A previous study showed that, after purification on a continuous gradient, the top interface (1.062-1.096 density range) was 50-95% islet cells with varying amounts of ductal and degranulated acinar tissue; the middle interface (1.096–1.11 density range) contained 1–15% islets, ductal, and degranulated acini; and the pellet was mostly well-granulated acinar tissue with less than 1% islets (1). Therefore, the cells in the top and middle layers were used in this study. The cells from the top and middle layers were stained by dithizone and the remnant islets were deleted by hand-picking under a dissecting microscope. The duct-rich population after hand-picking was then cultured at 37°C and 5% CO2 in several different media (Table 1). DMEM (high glucose), DMEM/F12, RPMI-1640 KnockOut™ DMEM, Knockout™ SR XenoFree, GlutaMAX-1, β-mercaptoethanol, nonessential amino acids (NEAA), and bFGF were purchased from Invitrogen (Carlsbad, CA). CMRL-1066 was purchased from Mediatech-Cellgro. Cholera toxin (CTx), insulin, transferrin, sodium selenite, bovine serum albumin (BSA), and keratinocyte growth factor (KGF) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from BioWest (Nuaille, France).
Culture Protocols
ITS: 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite; hES medium: Knockout™ DMEM + 15% Knockout™ SR XenoFree + 2 mM GlutaMAX-1,0.1 mM β-mercaptoethanol, 0.1 mM NEAA, and 8 ng/ml bFGF; MM1: Miami-defined media #1.
Cell Induction and Differentiation
For inducing cell differentiation, the cells were cultured in DMEM with 10% FBS, 10 nM exendin-4, 10 mM nicotinamide, 10 ng/ml KGF, 100 nM PDX-1 protein, and 100 nM BETA2/NeuroD protein for 2 weeks. For PDX-1 and BETA2/NeuroD protein, the cDNAs were amplified by PCR using appropriate linker primers and then subcloned into the NdeI and XhoI sites of pET21b(+) (Novagen, Madison, WI) using a ligation kit (TaKaRa, Tokyo, Japan). BL21 (DE3) cells containing the expression plasmids were grown at 37°C to an OD600 of 0.8. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.1 mmol/L, and the cells were then incubated for 12 h at 24°C. Cells were sonicated and the supernatants were recovered and applied to a column of Ni-nitrilotriacetic acid agarose (Invitrogen, San Diego, CA).
Semiquantitative RT-PCR
Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen, Valencia, CA). After quantifying RNA by spectrophotometry, 2.5 μg of RNA was heated at 85°C for 3 min and then reverse-transcribed into cDNA in a 25-μl solution containing 200 units of Superscript II RNase H-RT (Invitrogen), 50 ng random hexamers (Invitrogen), 160 μmol/L dNTP, and 10 nmol/L dithiothreitol. The reaction consisted of 10 min at 25°C, 60 min at 42°C, and 10 min at 95°C. Polymerization reactions were performed in a Perkin-Elmer 9700 Thermocycler with 3 μl cDNA (20 ng RNA equivalents), 160 μmol/L cold dNTPs, 10 pmol each appropriate oligonucleotide primer, 1.5 mmol/L MgCl2, and 5 units AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT). The oligonucleotide primers and cycle number used for semiquantitative PCR are shown in Table 2. The thermal cycle profile used a 10-min denaturing step at 94°C followed by amplification cycles (1 min denaturation at 94°C, 1 min annealing at 57°C, and 1 min extension at 72°C) with a final extension step of 10 min at 72°C. The steps taken to validate these measurements were previously reported (14).
List of Gene-Specific Primers
Results
Culture of Human Duct-Rich Population
Duct-rich populations from human pancreata were used in this study. After hand-picking islets from the top and middle layers under a dissecting microscope, the duct-rich population was cultured in two culture media. Protocol #1 was CMRL-1066 with 10% FBS and 100 ng/ml CTx for 3 days and then in DMEM/F12 with 100 ng/ml CTx, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 0.2% BSA, and 25 ng/ml KGF according to the previous report by Yamamoto et al. (Y1/Y2 medium) (32). Protocol #2 was DMEM + 20% FBS (BioWest Cat #S1560, Lot #SO5094S1560) according to our previous report (N1 medium) (19). However, we were unable to establish human pancreatic stem cell line using these culture protocols. Therefore, we prepared 23 kinds of culture protocols based on Y1/Y2 medium (32), N1 medium, or the medium for human embryonic stem cells (Table 1). The morphologies of duct-rich populations in each culture medium are shown in Figure 1. The cells cultured in #1, #7, and #8 media, which are “serum-free” Y1/Y2-like media, formed “cobblestone” morphologies, similar to mouse pancreatic stem cell lines. On the other hand, the cells cultured in #2–4, #10–15, and #20–23 media, which contain more than 1% FBS, formed “fibroblast-like” morphologies. The cells cultured in #9, which contains 0.5% FBS, displayed a mixed morphology that was part cobblestone and part fibroblast-like. The cells cultured in #6 and #16–19 media, which are similar to the medium for human ES cells, formed fibroblast-like morphologies and exhibited cell aggregation.
Morphology of the duct-rich population. The cells from the top and middle layers after islet purification were stained by dithizone and the remnant islets were deleted by hand-picking under a dissecting microscope. The duct-rich population, after removal of islets, was then cultured at 37°C and 5% CO2 in several media (Table 1). The cells were unable to attach to the flask in culture medium #5. Scale bar: 100 μm.
Gene Expression and Growth Activity of the Duct-Rich Population
We used the cells from culture conditions #1, #2, #4, #6, or #8 in the following experiments because protocols #1 and #2 are the culture conditions of mouse pancreatic stem cells reported by Yamamoto et al. (32) and our group (19), respectively; #4 was used as a representative of serum-containing media; #6 was used as a representative of culture media for human ES cells; and #8 was used as a representative of serum free media. The cells from 2-week cultures under culture conditions #1, #2, #4, #6, or #8 were examined for the mRNA expression of insulin, PDX-1, CK-19, and GLP-1 receptor by RT-PCR. All of these cells expressed PDX-1 and CK-19, markers for pancreatic ducts, but they did not express insulin mRNA (Fig. 2A). All of these cells also expressed glucagon-like peptide-1 (GLP-1) receptor (Fig. 2A). The cells under culture conditions #1, #2, #4, #6, or #8 were also examined for their growth activity. The cells divided actively for 30 days, the population doubling level (PDL) was 6–10 (because the cell number increased between 26 and 210 times). However, the cells stopped dividing after 30 days under all of culture conditions #1, #2, #4, #6, or #8 (Fig. 2B). Even in other culture conditions, the cells were unable to divide after 30 days (data not shown). During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence (data not shown).
Gene expression and growth activity of the duct-rich population. (A) Expression of insulin, PDX-1, CK-19, and GLP-1 receptor under #1, #2, #4, #6, or #8 culture conditions. The cells were examined for the mRNA expression of insulin, PDX-1, CK-19, and GLP-1 receptor by RT-PCR. (B) Growth activity of the duct-rich population under culture conditions #1, #2, #4, #6, or #8.
Cell Characterization Before and After Induction of Exendin-4 and Transduction of PDX-1 and BETA2/NeuroD Proteins
Although the cells from the duct-rich population under condition #1 expressed PDX-1 mRNA, which is one of the most crucial transcription factors for embryonic development of the mouse endocrine pancreas, insulin mRNA was not observed in the cells (Fig. 2A). On the other hand, other transcription factors such as BETA2/NeuroD, Pax6, and Isl-1 were not expressed in untreated cells. Transcripts encoding PDX-1, NeuroD, Pax6, and Isl-1 were abundant in adult human islets.
We previously reported that PDX-1 and BETA2/NeuroD protein transduction technology could be a safe and valuable strategy for facilitating the differentiation of stem/progenitor cells into insulin-producing cells without requiring gene transfer technology (13, 14, 19, 25, 26). The cells from a duct-rich population expressed the GLP-1 receptor, as shown in Figure 2A. Therefore, for inducing cell differentiation, the cells were cultured with exendin-4, PDX-1 protein, and BETA2/NeuroD protein for 2 weeks (Fig. 3A). The treated cells induced the expression of insulin mRNA, although the level seemed low compared to the level in primary human islets (Fig. 3B), and insulin in culture medium was undetectable (data not shown). We also examined the expression of other transcription factors and pancreas-related genes. After induction, BETA2/NeuroD, Pax6, and Isl-1 transcription factors were induced (Fig. 3B) but not GLUT2 or glucokinase (data not shown). These data suggest that the cells from a duct-rich population have a potential for differentiation into insulin-producing cells. The culture conditions induced insulin and pancreas-related gene expression in the cells, although the induced cells were still immature.
Cell characterization before and after induction of exendin-4 and transduction of PDX-1 and BETA2/NeuroD proteins. (A) Induction protocol. Duct-rich populations were cultured in culture medium #1 for 2 weeks and then cultured in DMEM with 10% FBS, 10 nM exendin-4, 10 mM nicotinamide, 10 ng/ml KGF, 100 nM PDX-1 protein, and 100 nM BETA2/NeuroD protein for 2 weeks. (B) Expression of pancreas-related genes in duct-rich populations before and after treatment of induction medium. PCR was performed in a Perkin-Elmer 9700 Thermocycler with 2 μl cDNA (20 ng RNA equivalent) from human cells. The oligonucleotide primers and cycle number used for semiquantitative PCR are shown in Table 2. Human islets were used as a positive control.
Discussion
This study demonstrates that the culture of human pancreatic progenitor cells could be differentiated into insulin-producing cells in vitro. Because there is limited in vitro growth of adult islet cells, the ability to cultivate islets in vitro from a duct-rich population that is usually discarded after islet isolation provides an important approach to β-cell replacement therapy. For inducing cell differentiation, we used exendin-4, PDX-1 protein, and BETA2/NeuroD protein. GLP-1/exendin-4 has incretin effects, enhancing insulin secretion. It also stimulates β-cell replication and neogenesis and has antiapoptotic effects (2). PDX-1 plays a central role in regulating pancreatic development and is associated with islet neogenesis and the differentiation of insulin-producing cells from progenitor cells in the adult pancreas (1, 7, 24). BETA2/NeuroD is also a key regulator of pancreatic islet morphogenesis and insulin gene transcription (8, 12). We previously reported that PDX-1 and BETA2/NeuroD proteins can permeate cells by the protein transduction domain sequences in their structures and that the proteins, transduced into cultures of pancreatic ducts, induce insulin gene expression (13, 14, 19, 25, 26). As shown in Figure 3B, 2 weeks after culture with exendin-4, PDX-1 protein, and BETA2/NeuroD protein, the cells induced insulin mRNA. However, the level of insulin mRNA was low compared with primary human islets. Insulin in culture medium was undetectable, suggesting that the induced cells were still immature. Therefore, further optimization of conditions is needed to generate a sufficient yield of insulin-producing cells for transplantation in diabetes.
We previously reported that mouse pancreatic stem cells could be maintained by repeated passages for more than 1 year without growth inhibition (19). However, the human cells from the duct-rich population were unable to divide after 30 days under any culture conditions in this study. There are some possibilities for these differences. One possibility is that the human pancreatic stem cells were unable to maintain an undifferentiated state under any culture conditions in this study. Indeed, the culture conditions of embryonic stem cells are different between human and mouse. If so, we need to evaluate other culture conditions. Another possibility is that pancreas preservation and/or isolation stress affected the survival of human pancreatic stem cells because human islet isolation is likely more stressful for cells than mouse islet isolation. A third possibility is that there are no stem cells in the human pancreas, while there are some stem cells in the mouse pancreas. However, there may be no stem cells in both human and mouse pancreata, indicating our mouse pancreatic stem cells may be due to spontaneously “immortalized” cells. Our mouse pancreatic stem cells were maintained only in a specific culture condition; they do not have tumorigenic properties, and do have a normal chromosome. Therefore, we believe that there are at least some stem cells in the mouse pancreas.
We evaluated human pancreatic progenitor cells from a duct-rich population. Replacement of the β-cell mass offers an alternative to standard insulin treatment and may overcome the long-term side effects associated with current therapies. Our observations highlight the important hurdles to be overcome to make β-cell replacement therapy available to a larger number of people with type 1 and 2 diabetes mellitus.
Footnotes
Acknowledgments
The authors wish to thank Dr. Carson Harrod for editing the manuscript and Ms. Yoshiko Tamura for technical support. This work was supported in part by the Juvenile Diabetes Research Foundation International (JDRFI), Otsuka Pharmaceutical Factory, Inc., and the All Saints Health Foundation.