Adult Pancreas Generates Multipotent Stem Cells and Pancreatic and Nonpancreatic Progeny
http://www.100md.com
《干细胞学杂志》
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Key Words. Stem cells ? Pancreatic islet ? Differentiation ? Multipotent stem cells ? Diabetes
Correspondence: Nadya Lumelsky, Ph.D., Islet and Autoimmunity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, CRC, Room 5-5940, 10 Center Drive, Bethesda, Maryland 20892-1453, USA. Telephone: 301-451-9834; Fax: 301-480-1269; e-mail: nadyal@intra.niddk.nih.gov
ABSTRACT
Insulin injections alleviate hyperglycemia in most patients with diabetes. However, they do not provide dynamic control of glucose homeostasis. Consequently, patients with long-term diabetes commonly develop life-threatening complications such as cardiovascular and kidney disease, neuropathy, and blindness. It has recently been shown that sustained independence from insulin injections can be achieved by transplantation of pancreatic islets into patients with diabetes . Unfortunately, practical application of this clinical protocol is severely hampered by the shortage of islets available for transplantation. If functional ?-cells and islets could be generated ex vivo, present severe islet shortage could be overcome. Another possible approach for restoration of islet cell mass is enhancement of endogenous regenerative capacity of endocrine pancreas. Recent results in rodents and humans suggest that pancreas has an extensive capacity to regenerate after injury . In fact, it has been hypothesized that diabetes might result from ?-cell destruction overpowering ?-cell regeneration and that even a long time after the onset of the disease, ?-cell regeneration might still take place . It thus might be possible to restore ?-cell mass in patients with diabetes by shifting the equilibrium between regeneration and destruction in the direction of regeneration.
Many adult mammalian organs contain a reservoir of tissue-specific stem and progenitor cells . Throughout the life of an organism, stem and progenitor cells are used for replenishing the differentiated cell mass to compensate for cell loss during normal cell turnover and after organ damage. Although it is thought that cell replacement occurs primarily via differentiation of pre-existing resident organ-specific stem and progenitor cells, there are some notable exceptions. For example, it has been shown that adult mammalian liver can efficiently regenerate as a result of proliferation of mature hepatocytes . Also, in lower vertebrates, limb regeneration after injury occurs via dedifferentiation of mature tissue into transient stem cells, which expand and redifferentiate to regenerate the missing limb . Whether similar transitions occur in higher vertebrates remains to be determined.
During embryogenesis, the pancreas is derived from the endoderm . Neural cells, on the other hand, are derived from ectoderm. Despite their different embryological origins, pancreatic and neural cells express many common enzymes and other markers and have similarities in developmental control mechanisms . An intermediate filament protein, nestin, a marker of neural stem and progenitor cells , is also expressed in pancreas. Recently it has been proposed that nestin may mark pancreatic stem or progenitor cells . This claim was challenged, however, by others studies that suggest that nestin is expressed in pancreatic mesenchyme and not in the epithelium from which pancreatic endocrine cells are thought to originate .
It has been argued that in the mammalian pancreas, new hormone-producing cells arise either through islet cell replication or through differentiation from progenitor and stem cells via a process called islet neogenesis . Because new islets often arise in close proximity to pancreatic ducts, it has been suggested that pancreatic stem or progenitor cells might reside within the ducts . However, recent results do not support this hypothesis . Rigorous testing of the origins of cells that can give rise to new islet cells is complicated by the lack of specific markers for the putative pancreatic stem and progenitor cells and by the difficulty of following individual cells’fate in the pancreas. An in vitro differentiation system allowing generation of new pancreatic endocrine cells would greatly benefit this work .
In this study we present a new in vitro differentiation system derived from adult mouse islet-enriched fractions (IEFs). When IEFs are cultured on adhesive substrates, they first generate a heterogeneous population of proliferating cells. After aggregation, the IEF-derived cells form three-dimensional islet-like cell clusters (ILCCs), which produce insulin/C-peptide and other islet endocrine hormones, glucagons, and somatostatin. Our results show that new hormone-producing cells are actively generated in this in vitro system. Moreover, we show that embryonic-like cells exhibiting neural and stem cell properties arise in the IEF cultures. These findings uncover a previously unrecognized capacity of adult pancreas to acquire embryonic phenotype and to generate pancreatic and nonpancreatic progeny.
MATERIALS AND METHODS
ILCCs Exhibit Phenotype of Rodent Islets
To examine the potential of adult mouse pancreas to generate new hormone-producing cells, we cultured IEFs using a modification of protocols previously shown to promote neural differentiation of central nervous system (CNS) stem cells and pancreatic differentiation of embryonic stem cells . When freshly isolated IEFs are cultured on fibronectin in fetal bovine serum–free medium, in the presence of bFGF and LIF, they lose their three-dimensional architecture and generate morphologically heterogeneous rapidly proliferating cell populations (Fig. 1A; also see Materials and Methods for detailed description of the culturing protocol). Notably, cells with neural-like morphology are a common feature of stage 1 cultures (see below). When the cells are replated at high density on laminin-1 at the end of stage 1, they gradually aggregate to form three-dimensional ILCCs. If, however, the same cells are replated at low density on either laminin-1 or fibronectin or at high density on fibronectin, the ILCC formation is inefficient (Figs. 1B, 1C). If the cells are not replated at the end of stage 1 but are continually maintained on fibronectin, no ILCC formation is observed. Instead, neural-like cells become a predominant cell type (unpublished data).
To quantify the effect of different culture conditions on ILCC differentiation, we compared intracellular C-peptide concentration after culturing stage 2 cells at high versus low density and on laminin-1 versus fibronectin. We chose to measure C-peptide rather than insulin to eliminate a potential contribution to the signal from the bovine insulin present in the culture media . Because, as a result of proteolytic processing of proinsulin, insulin and C-peptide are generated in equimolar quantities, the C-peptide production is a reflection of insulin production. When measured at the end of the culture in two independent experiments, the concentration of C-peptide in high-density laminin-1 cultures is 2- to 3.5-times higher than its concentration in high-density fibronectin cultures (Fig. 1B). If the cells are cultured on fibronectin at low density, the C-peptide concentration drops an additional 2- to 10-fold. Furthermore, when cells are cultured at low density on laminin, the concentration of C-peptide is reduced two to five times compared with cultures at high density on laminin. These results suggest that both cell–cell and cell–extracellular matrix interactions may be playing a functional role in this differentiation system. Given our finding that high-density laminin-1 cultures result in the best outcome, we used these conditions for all of the subsequent experiments described below.
To investigate the dynamics of C-peptide production during the culture, we compared intracellular C-peptide concentration per microgram of protein and total intracellular C-peptide content of fresh IEFs with those of cultured IEFs at the end of stages 1 and 3 (the latter value reflects the amount of C-peptide present in the whole-cell preparation at a given stage of culture). The results of three independent experiments (Figs. 1D, 1E) demonstrate that at the end of stage 1, the cultures become largely depleted of C-peptide. Our results suggest that increase in the rate of apoptosis as well as reduced hormone production by the IEFs is responsible for the observed C-peptide depletion of late stage 1 cultures (see below). The reduction in C-peptide level is followed by its increase at the end of the culture.
Immunocytochemical analysis of C-peptide and ? cell–specific transcription factor PDX-1 shows that, similar to native ? cells, C-peptide+ cells exhibit nuclear localization of PDX-1 (Fig. 1C, right side). Using immunocytochemistry, we have additionally found that other islet hormones, glucagon and somatostatin, are also produced by the ILCCs (unpublished data and Fig. 3B). Our analysis of functional maturation of the ILCCs shows that they release C-peptide in response to glucose with kinetics characteristic of normal islets (Fig. 1F). At 16.7 mM glucose, the release of C-peptide is nearly fivefold higher than that at the baseline, at 5 mM glucose.
Figure 3. New hormone cells are generated in IEF cultures. Each row shows split images of the same microscopic field. Left, immunostaining for the markers, as indicated; right, nuclear DAPI staining. (A): Immunocytochemical analysis of C-peptide/BrdU 24-hour pulse experiment. Several C-peptide+/BrdU+ cells and their corresponding nuclei on the left are marked with arrowheads. Scale bar = 20 μm (Zeiss Axiovert 2 Plus). (B): BrdU pulse chase analysis of endocrine hormone-producing cells. Top, general scheme of BrdU pulse chase analysis. Immunocytochemical analysis of hormone+/BrdU+ cells. Several hormone+/BrdU+ cells and their corresponding nuclei on the right are marked with arrowheads (Zeiss LSM510). Scale bar = 20 μm. Abbreviations: BrdU, bromodeoxyurindine; IEF, islet-enriched fraction; IHC, immunohistochemistry.
Expression of islet hormone-specific mRNAs during different stages of culture development was analyzed by RT-PCR (Fig. 2). In contrast to the C-peptide levels, the steady-state levels of these mRNAs do not change significantly during the culture period. Enumeration of C-peptide, glucagon, and somatostatin-expressing cells (Tables 1A and 1B) shows that the percentage of C-peptide–expressing cells in the culture increases between the end of stages 1 and 3. Also, at the end of stage 3, the relative ratios of different hormone-expressing cells approximate those of the rodent islets. In summary, these results demonstrate that ILCCs have phenotypic characteristics similar to those of native islets.
Table 1A. Enumeration of endocrine hormone-producing cells
Table 1B. Enumeration of BrdU+ cells within endocrine- and neural-like cell populations
Extensive Cell Proliferation and Apoptosis Take Place during Stage 1
We next investigated the extent of contribution of the input IEF cell pool to the ILCCs obtained at the end of the culture. To this end, we measured cellular proliferation and apoptosis during the course of the IEF cultures. For cell proliferation analysis, the cells were pulsed for 6 hours with thymidine analog, BrdU, which incorporates into DNA of the replicating cells. After the pulse, the cells were fixed, and the incorporated BrdU was detected using immunocytochemistry with BrdU-specific antibody. Quantitative analysis of BrdU incorporation (Fig. 2A) demonstrates that cells in the IEF cultures proliferate throughout the culturing period. The peak of proliferation occurs between days 4 and 6 of stage 1. During this time window, 40%–50% of all cells in the culture become BrdU positive after 6h pulse. Most of the proliferating cells are found either in or around the flattened cells clusters, which lost their three-dimensional architecture. Occasionally we find proliferating cells at the periphery of three-dimensional clusters. Only few proliferating cells are detected within an interior of the three-dimensional clusters (Fig. 2A, right side of the panel; also see Fig. 2A).
The level of apoptosis was estimated using Caspase-GloTM luminescent assay (Promega), which measures caspase-3 and caspase-7 activity in cell lysates. Caspase-3 and -7 proteases are key effectors of apoptosis in mammalian cells . The results (Fig. 2B) show that the input IEF population contains a large proportion of apoptotic cells. Also, early stage 1 cultures undergo active apoptosis. Moreover, we found that in early stage 1 cultures, a large fraction of C-peptide–expressing cells coexpress caspase 3 (Fig. 2B, right side of the panel). Overall, the results of BrdU incorporation and apoptosis analysis demonstrate that cell proliferation accompanied by extensive apoptosis takes place during stage 1 of IEF culture. They suggest that stage 3 cultures become considerably depleted of the input IEF cells.
New Islet-Like Cells Are Generated in IEF Cultures
To examine if new C-peptide/insulin-producing cells are generated during the course of IEF culture, we carried out BrdU pulse and BrdU pulse chase analysis of C-peptide–expressing cells. When we pulsed stage 1 cultures with BrdU for 2 hours, we could not detect any double-labeled C-peptide+/BrdU+ cells (unpublished results). However, when the BrdU pulse was extended to 24 hours, both C-peptide+/ BrdU+ and insulin+/BrdU+ cells were detected (Fig. 3A and unpublished data). These double-labeled cells appear primarily within flattened epithelial clusters, which are a common feature of early stage 1 cultures. These double-labeled cells are often found at the clusters’ periphery, and they appear to exhibit a reduced immunoreactivity to C-peptide antibody. These results indicate that ? cells may be losing C-peptide expression as they divide in culture.
The scheme of BrdU pulse chase analysis is shown at the top of Figure 3B. The cells were pulsed with BrdU during stage 1 for 24 hours and then were maintained without BrdU for the remainder of the experiment. The results of this experiment (Fig. 3B) demonstrate that a subpopulation of stage 3 C-peptide, glucagon, and somatostatin-expressing cells is derived from the proliferating cells. In particular, our quantitative analysis shows that between 20% and 40% of stage 3 hormone-expressing cells are derived from proliferating cells (Tables 1A and 1B). We obtained similar results in at least three independent experiments. In summary, the results of BrdU pulse and BrdU pulse chase analysis indicate that new hormone-producing cells are generated during the course of the IEF cultures.
Stage 1 Cultures Express a Wide Range of Embryonic Neural and Stem Cell Genes
As described above, we observe morphologically heterogeneous neural-like cells in stage 1 cultures. We decided to examine if indeed these cells express neural-specific markers. In addition to the neural progenitor cell marker, nestin, we examined a marker of astrocytes, GFAP ; a marker of early neurons, neuronal-specific ? III subunit of ?-tubulin recognized by TUJ1 antibody ; a marker of embryonic neural crest, transcription factor Sox10 ; and a marker of oligodendrocytic progenitors, O4 . We also examined expression of neuroepithelial and radial glial cell marker of embryonic CNS, RC-2 . Notably, in the embryonic brain, radial glia is thought to possess stem cell properties . The results of immunocytochemical analysis (Fig. 4) show that all the examined markers are abundantly expressed at the end of stage 1 in a partially overlapping pattern. For example, we detect distinct subpopulations of nestin+ cells expressing TUJ1 and GFAP. Most O4+ cells found within cell clusters do not coexpress GFAP. We found that, similar to adult brain , adult IEFs do not express RC-2 (unpublished data). In contrast, an abundant population of RC-2+/nestin+ cells is found in late stage 1 cultures (Figs. 4E, 4F). Reminiscently of their arrangement in the embryonic brain, these cells are organized in parallel cell bundles. Moreover, similarly to mouse CNS, most RC-2+ cells do not express GFAP (Fig. 4F). The results of BrdU lineage tracing analysis show that most TUJ1, RC-2, nestin, and GFAP cells are derived from proliferating cell populations (Table 1B) and thus are generated de novo during the course of the culture.
Figure 4. IEF cultures exhibit neural phenotype. (A–F): Expression of neural markers in day-10 IEF cultures. Image in (A) was obtained with Zeiss Axiovert 2 Plus; images in (B), (C), (D), (E), and (F) were obtained with Zeiss LSM510. Scale bars = 50 μm. Abbreviations: GFAP, glial fibrillary acidic protein; IEF, islet-enriched fraction.
Neural and stem cell phenotype of IEF cultures was analyzed additionally by RT-PCR (Fig. 5). The results demonstrate that expression of GFAP, nestin, and astrocytic marker, Ca2+ binding protein S100? , peaks at the end of stage 1 and falls at the end of the culture. This pattern of expression was also confirmed by immunocytochemistry (unpublished data). Having found neural embryonic-like cells in these adult pancreatic cultures, we decided to expand the RT-PCR analysis to a wider panel of stem cell–specific and other early developmental genes (Table 2). Our results show that by the end of stage 1, the IEF cultures initiate expression of many genes associated with embryonic and stem cell phenotype. For example, we detect a marker of side population, the ABC pump Bcrp1/Abcg2 , which is found in a variety of multipotent stem cell types. Although the day 0 cDNA preparation used for the RT-PCR analysis shown in Figure 5 is negative for Bcrp1/Abcg2, we detected Bcrp1/Abcg2 signal in other day-0 cDNA preparations. Further, Bmi1, a polycomb-group transcriptional repressor required for the maintenance of adult stem cells in several tissues , is upregulated during stage 1. Interestingly, we also detect the Bmi1 signal in IEFs before culture initiation. To our knowledge, expression of Bmi1 in adult pancreas has not been previously documented.
Figure 5. IEF cultures express a wide range pancreatic, neural, and stem cell–specific genes. Time-course RT-PCR analysis of pancreatic, neural, and stem cell gene expression. Negative control PCR amplifications, "RT (day 10)," were carried out with mock cDNA samples, which were prepared with day-10 RNA, omitting RT from the cDNA reaction mixture. The results in the two panels shown were obtained with two independent RNA time courses. Similar results for the markers shown were obtained in at least three independents experiments. Abbreviations:GFAP, glial fibrillary acidic protein; IEF, islet-enriched fraction; RT-PCR, reverse transcriptase–polymerase chain reaction.
Table 2. Neural and stem cell markers shown in Figure 5
Transient induction of markers of ES cells/preimplantation embryo, Oct-4, nanog, Sox2, and FoxD3 is particularly intriguing. It suggests that a short-lived ES-like sub-population may be present in late-stage 1 IEF cultures. Because, in addition to expression in the embryonic stem cells, Sox2 and FoxD3 are also expressed in embryonic neural crest, perhaps a portion of Sox2 and FoxD3 signal can be attributed to a neural crest–like cell population that arises in the IEF cultures. In fact, we have found that other neural crest–specific mRNAs, Twist, Snail, Slug, Sip1, and Sox10 (see also Fig. 4C) , are induced during stage 1 (Fig. 5). We reproducibly observed a similar mRNA expression pattern for all of the examined markers in different independent IEF in vitro culture experiments.
DISCUSSION
We thank David Harlan, Marvin Gershengorn, and Derek LeRoith for their comments on the manuscript and Arnold Kriegstein for helpful discussions. We are grateful to Ron McKay for the gift of anti-nestin antibody, Chris Wright and Joel Habener for the gifts of anti-PDX-1 antibodies, and Bill Pavan for anti-Sox10 antibody. We thank Oksana Gavrilova and Stephanie Pack for their help with IEF isolations, Carolyn Smith for her help with laser scanning confocal microscopy, and Cheol Hong Park and Kristina Buac for their help with cell culture and preparation of the Figures.
A work supporting our finding of neural differentiation capacity of adult mouse pancreas (Seaberg, R.M. et al. Clonal Identification of Multipotent Precursors from Adult Mouse Pancreas that Generate Neural and Pancreatic Lineages. Nature Biotechnology 22, 1115–1124, 2004) had appeared in print after our manuscript was accepted for pulication.
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Key Words. Stem cells ? Pancreatic islet ? Differentiation ? Multipotent stem cells ? Diabetes
Correspondence: Nadya Lumelsky, Ph.D., Islet and Autoimmunity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 10, CRC, Room 5-5940, 10 Center Drive, Bethesda, Maryland 20892-1453, USA. Telephone: 301-451-9834; Fax: 301-480-1269; e-mail: nadyal@intra.niddk.nih.gov
ABSTRACT
Insulin injections alleviate hyperglycemia in most patients with diabetes. However, they do not provide dynamic control of glucose homeostasis. Consequently, patients with long-term diabetes commonly develop life-threatening complications such as cardiovascular and kidney disease, neuropathy, and blindness. It has recently been shown that sustained independence from insulin injections can be achieved by transplantation of pancreatic islets into patients with diabetes . Unfortunately, practical application of this clinical protocol is severely hampered by the shortage of islets available for transplantation. If functional ?-cells and islets could be generated ex vivo, present severe islet shortage could be overcome. Another possible approach for restoration of islet cell mass is enhancement of endogenous regenerative capacity of endocrine pancreas. Recent results in rodents and humans suggest that pancreas has an extensive capacity to regenerate after injury . In fact, it has been hypothesized that diabetes might result from ?-cell destruction overpowering ?-cell regeneration and that even a long time after the onset of the disease, ?-cell regeneration might still take place . It thus might be possible to restore ?-cell mass in patients with diabetes by shifting the equilibrium between regeneration and destruction in the direction of regeneration.
Many adult mammalian organs contain a reservoir of tissue-specific stem and progenitor cells . Throughout the life of an organism, stem and progenitor cells are used for replenishing the differentiated cell mass to compensate for cell loss during normal cell turnover and after organ damage. Although it is thought that cell replacement occurs primarily via differentiation of pre-existing resident organ-specific stem and progenitor cells, there are some notable exceptions. For example, it has been shown that adult mammalian liver can efficiently regenerate as a result of proliferation of mature hepatocytes . Also, in lower vertebrates, limb regeneration after injury occurs via dedifferentiation of mature tissue into transient stem cells, which expand and redifferentiate to regenerate the missing limb . Whether similar transitions occur in higher vertebrates remains to be determined.
During embryogenesis, the pancreas is derived from the endoderm . Neural cells, on the other hand, are derived from ectoderm. Despite their different embryological origins, pancreatic and neural cells express many common enzymes and other markers and have similarities in developmental control mechanisms . An intermediate filament protein, nestin, a marker of neural stem and progenitor cells , is also expressed in pancreas. Recently it has been proposed that nestin may mark pancreatic stem or progenitor cells . This claim was challenged, however, by others studies that suggest that nestin is expressed in pancreatic mesenchyme and not in the epithelium from which pancreatic endocrine cells are thought to originate .
It has been argued that in the mammalian pancreas, new hormone-producing cells arise either through islet cell replication or through differentiation from progenitor and stem cells via a process called islet neogenesis . Because new islets often arise in close proximity to pancreatic ducts, it has been suggested that pancreatic stem or progenitor cells might reside within the ducts . However, recent results do not support this hypothesis . Rigorous testing of the origins of cells that can give rise to new islet cells is complicated by the lack of specific markers for the putative pancreatic stem and progenitor cells and by the difficulty of following individual cells’fate in the pancreas. An in vitro differentiation system allowing generation of new pancreatic endocrine cells would greatly benefit this work .
In this study we present a new in vitro differentiation system derived from adult mouse islet-enriched fractions (IEFs). When IEFs are cultured on adhesive substrates, they first generate a heterogeneous population of proliferating cells. After aggregation, the IEF-derived cells form three-dimensional islet-like cell clusters (ILCCs), which produce insulin/C-peptide and other islet endocrine hormones, glucagons, and somatostatin. Our results show that new hormone-producing cells are actively generated in this in vitro system. Moreover, we show that embryonic-like cells exhibiting neural and stem cell properties arise in the IEF cultures. These findings uncover a previously unrecognized capacity of adult pancreas to acquire embryonic phenotype and to generate pancreatic and nonpancreatic progeny.
MATERIALS AND METHODS
ILCCs Exhibit Phenotype of Rodent Islets
To examine the potential of adult mouse pancreas to generate new hormone-producing cells, we cultured IEFs using a modification of protocols previously shown to promote neural differentiation of central nervous system (CNS) stem cells and pancreatic differentiation of embryonic stem cells . When freshly isolated IEFs are cultured on fibronectin in fetal bovine serum–free medium, in the presence of bFGF and LIF, they lose their three-dimensional architecture and generate morphologically heterogeneous rapidly proliferating cell populations (Fig. 1A; also see Materials and Methods for detailed description of the culturing protocol). Notably, cells with neural-like morphology are a common feature of stage 1 cultures (see below). When the cells are replated at high density on laminin-1 at the end of stage 1, they gradually aggregate to form three-dimensional ILCCs. If, however, the same cells are replated at low density on either laminin-1 or fibronectin or at high density on fibronectin, the ILCC formation is inefficient (Figs. 1B, 1C). If the cells are not replated at the end of stage 1 but are continually maintained on fibronectin, no ILCC formation is observed. Instead, neural-like cells become a predominant cell type (unpublished data).
To quantify the effect of different culture conditions on ILCC differentiation, we compared intracellular C-peptide concentration after culturing stage 2 cells at high versus low density and on laminin-1 versus fibronectin. We chose to measure C-peptide rather than insulin to eliminate a potential contribution to the signal from the bovine insulin present in the culture media . Because, as a result of proteolytic processing of proinsulin, insulin and C-peptide are generated in equimolar quantities, the C-peptide production is a reflection of insulin production. When measured at the end of the culture in two independent experiments, the concentration of C-peptide in high-density laminin-1 cultures is 2- to 3.5-times higher than its concentration in high-density fibronectin cultures (Fig. 1B). If the cells are cultured on fibronectin at low density, the C-peptide concentration drops an additional 2- to 10-fold. Furthermore, when cells are cultured at low density on laminin, the concentration of C-peptide is reduced two to five times compared with cultures at high density on laminin. These results suggest that both cell–cell and cell–extracellular matrix interactions may be playing a functional role in this differentiation system. Given our finding that high-density laminin-1 cultures result in the best outcome, we used these conditions for all of the subsequent experiments described below.
To investigate the dynamics of C-peptide production during the culture, we compared intracellular C-peptide concentration per microgram of protein and total intracellular C-peptide content of fresh IEFs with those of cultured IEFs at the end of stages 1 and 3 (the latter value reflects the amount of C-peptide present in the whole-cell preparation at a given stage of culture). The results of three independent experiments (Figs. 1D, 1E) demonstrate that at the end of stage 1, the cultures become largely depleted of C-peptide. Our results suggest that increase in the rate of apoptosis as well as reduced hormone production by the IEFs is responsible for the observed C-peptide depletion of late stage 1 cultures (see below). The reduction in C-peptide level is followed by its increase at the end of the culture.
Immunocytochemical analysis of C-peptide and ? cell–specific transcription factor PDX-1 shows that, similar to native ? cells, C-peptide+ cells exhibit nuclear localization of PDX-1 (Fig. 1C, right side). Using immunocytochemistry, we have additionally found that other islet hormones, glucagon and somatostatin, are also produced by the ILCCs (unpublished data and Fig. 3B). Our analysis of functional maturation of the ILCCs shows that they release C-peptide in response to glucose with kinetics characteristic of normal islets (Fig. 1F). At 16.7 mM glucose, the release of C-peptide is nearly fivefold higher than that at the baseline, at 5 mM glucose.
Figure 3. New hormone cells are generated in IEF cultures. Each row shows split images of the same microscopic field. Left, immunostaining for the markers, as indicated; right, nuclear DAPI staining. (A): Immunocytochemical analysis of C-peptide/BrdU 24-hour pulse experiment. Several C-peptide+/BrdU+ cells and their corresponding nuclei on the left are marked with arrowheads. Scale bar = 20 μm (Zeiss Axiovert 2 Plus). (B): BrdU pulse chase analysis of endocrine hormone-producing cells. Top, general scheme of BrdU pulse chase analysis. Immunocytochemical analysis of hormone+/BrdU+ cells. Several hormone+/BrdU+ cells and their corresponding nuclei on the right are marked with arrowheads (Zeiss LSM510). Scale bar = 20 μm. Abbreviations: BrdU, bromodeoxyurindine; IEF, islet-enriched fraction; IHC, immunohistochemistry.
Expression of islet hormone-specific mRNAs during different stages of culture development was analyzed by RT-PCR (Fig. 2). In contrast to the C-peptide levels, the steady-state levels of these mRNAs do not change significantly during the culture period. Enumeration of C-peptide, glucagon, and somatostatin-expressing cells (Tables 1A and 1B) shows that the percentage of C-peptide–expressing cells in the culture increases between the end of stages 1 and 3. Also, at the end of stage 3, the relative ratios of different hormone-expressing cells approximate those of the rodent islets. In summary, these results demonstrate that ILCCs have phenotypic characteristics similar to those of native islets.
Table 1A. Enumeration of endocrine hormone-producing cells
Table 1B. Enumeration of BrdU+ cells within endocrine- and neural-like cell populations
Extensive Cell Proliferation and Apoptosis Take Place during Stage 1
We next investigated the extent of contribution of the input IEF cell pool to the ILCCs obtained at the end of the culture. To this end, we measured cellular proliferation and apoptosis during the course of the IEF cultures. For cell proliferation analysis, the cells were pulsed for 6 hours with thymidine analog, BrdU, which incorporates into DNA of the replicating cells. After the pulse, the cells were fixed, and the incorporated BrdU was detected using immunocytochemistry with BrdU-specific antibody. Quantitative analysis of BrdU incorporation (Fig. 2A) demonstrates that cells in the IEF cultures proliferate throughout the culturing period. The peak of proliferation occurs between days 4 and 6 of stage 1. During this time window, 40%–50% of all cells in the culture become BrdU positive after 6h pulse. Most of the proliferating cells are found either in or around the flattened cells clusters, which lost their three-dimensional architecture. Occasionally we find proliferating cells at the periphery of three-dimensional clusters. Only few proliferating cells are detected within an interior of the three-dimensional clusters (Fig. 2A, right side of the panel; also see Fig. 2A).
The level of apoptosis was estimated using Caspase-GloTM luminescent assay (Promega), which measures caspase-3 and caspase-7 activity in cell lysates. Caspase-3 and -7 proteases are key effectors of apoptosis in mammalian cells . The results (Fig. 2B) show that the input IEF population contains a large proportion of apoptotic cells. Also, early stage 1 cultures undergo active apoptosis. Moreover, we found that in early stage 1 cultures, a large fraction of C-peptide–expressing cells coexpress caspase 3 (Fig. 2B, right side of the panel). Overall, the results of BrdU incorporation and apoptosis analysis demonstrate that cell proliferation accompanied by extensive apoptosis takes place during stage 1 of IEF culture. They suggest that stage 3 cultures become considerably depleted of the input IEF cells.
New Islet-Like Cells Are Generated in IEF Cultures
To examine if new C-peptide/insulin-producing cells are generated during the course of IEF culture, we carried out BrdU pulse and BrdU pulse chase analysis of C-peptide–expressing cells. When we pulsed stage 1 cultures with BrdU for 2 hours, we could not detect any double-labeled C-peptide+/BrdU+ cells (unpublished results). However, when the BrdU pulse was extended to 24 hours, both C-peptide+/ BrdU+ and insulin+/BrdU+ cells were detected (Fig. 3A and unpublished data). These double-labeled cells appear primarily within flattened epithelial clusters, which are a common feature of early stage 1 cultures. These double-labeled cells are often found at the clusters’ periphery, and they appear to exhibit a reduced immunoreactivity to C-peptide antibody. These results indicate that ? cells may be losing C-peptide expression as they divide in culture.
The scheme of BrdU pulse chase analysis is shown at the top of Figure 3B. The cells were pulsed with BrdU during stage 1 for 24 hours and then were maintained without BrdU for the remainder of the experiment. The results of this experiment (Fig. 3B) demonstrate that a subpopulation of stage 3 C-peptide, glucagon, and somatostatin-expressing cells is derived from the proliferating cells. In particular, our quantitative analysis shows that between 20% and 40% of stage 3 hormone-expressing cells are derived from proliferating cells (Tables 1A and 1B). We obtained similar results in at least three independent experiments. In summary, the results of BrdU pulse and BrdU pulse chase analysis indicate that new hormone-producing cells are generated during the course of the IEF cultures.
Stage 1 Cultures Express a Wide Range of Embryonic Neural and Stem Cell Genes
As described above, we observe morphologically heterogeneous neural-like cells in stage 1 cultures. We decided to examine if indeed these cells express neural-specific markers. In addition to the neural progenitor cell marker, nestin, we examined a marker of astrocytes, GFAP ; a marker of early neurons, neuronal-specific ? III subunit of ?-tubulin recognized by TUJ1 antibody ; a marker of embryonic neural crest, transcription factor Sox10 ; and a marker of oligodendrocytic progenitors, O4 . We also examined expression of neuroepithelial and radial glial cell marker of embryonic CNS, RC-2 . Notably, in the embryonic brain, radial glia is thought to possess stem cell properties . The results of immunocytochemical analysis (Fig. 4) show that all the examined markers are abundantly expressed at the end of stage 1 in a partially overlapping pattern. For example, we detect distinct subpopulations of nestin+ cells expressing TUJ1 and GFAP. Most O4+ cells found within cell clusters do not coexpress GFAP. We found that, similar to adult brain , adult IEFs do not express RC-2 (unpublished data). In contrast, an abundant population of RC-2+/nestin+ cells is found in late stage 1 cultures (Figs. 4E, 4F). Reminiscently of their arrangement in the embryonic brain, these cells are organized in parallel cell bundles. Moreover, similarly to mouse CNS, most RC-2+ cells do not express GFAP (Fig. 4F). The results of BrdU lineage tracing analysis show that most TUJ1, RC-2, nestin, and GFAP cells are derived from proliferating cell populations (Table 1B) and thus are generated de novo during the course of the culture.
Figure 4. IEF cultures exhibit neural phenotype. (A–F): Expression of neural markers in day-10 IEF cultures. Image in (A) was obtained with Zeiss Axiovert 2 Plus; images in (B), (C), (D), (E), and (F) were obtained with Zeiss LSM510. Scale bars = 50 μm. Abbreviations: GFAP, glial fibrillary acidic protein; IEF, islet-enriched fraction.
Neural and stem cell phenotype of IEF cultures was analyzed additionally by RT-PCR (Fig. 5). The results demonstrate that expression of GFAP, nestin, and astrocytic marker, Ca2+ binding protein S100? , peaks at the end of stage 1 and falls at the end of the culture. This pattern of expression was also confirmed by immunocytochemistry (unpublished data). Having found neural embryonic-like cells in these adult pancreatic cultures, we decided to expand the RT-PCR analysis to a wider panel of stem cell–specific and other early developmental genes (Table 2). Our results show that by the end of stage 1, the IEF cultures initiate expression of many genes associated with embryonic and stem cell phenotype. For example, we detect a marker of side population, the ABC pump Bcrp1/Abcg2 , which is found in a variety of multipotent stem cell types. Although the day 0 cDNA preparation used for the RT-PCR analysis shown in Figure 5 is negative for Bcrp1/Abcg2, we detected Bcrp1/Abcg2 signal in other day-0 cDNA preparations. Further, Bmi1, a polycomb-group transcriptional repressor required for the maintenance of adult stem cells in several tissues , is upregulated during stage 1. Interestingly, we also detect the Bmi1 signal in IEFs before culture initiation. To our knowledge, expression of Bmi1 in adult pancreas has not been previously documented.
Figure 5. IEF cultures express a wide range pancreatic, neural, and stem cell–specific genes. Time-course RT-PCR analysis of pancreatic, neural, and stem cell gene expression. Negative control PCR amplifications, "RT (day 10)," were carried out with mock cDNA samples, which were prepared with day-10 RNA, omitting RT from the cDNA reaction mixture. The results in the two panels shown were obtained with two independent RNA time courses. Similar results for the markers shown were obtained in at least three independents experiments. Abbreviations:GFAP, glial fibrillary acidic protein; IEF, islet-enriched fraction; RT-PCR, reverse transcriptase–polymerase chain reaction.
Table 2. Neural and stem cell markers shown in Figure 5
Transient induction of markers of ES cells/preimplantation embryo, Oct-4, nanog, Sox2, and FoxD3 is particularly intriguing. It suggests that a short-lived ES-like sub-population may be present in late-stage 1 IEF cultures. Because, in addition to expression in the embryonic stem cells, Sox2 and FoxD3 are also expressed in embryonic neural crest, perhaps a portion of Sox2 and FoxD3 signal can be attributed to a neural crest–like cell population that arises in the IEF cultures. In fact, we have found that other neural crest–specific mRNAs, Twist, Snail, Slug, Sip1, and Sox10 (see also Fig. 4C) , are induced during stage 1 (Fig. 5). We reproducibly observed a similar mRNA expression pattern for all of the examined markers in different independent IEF in vitro culture experiments.
DISCUSSION
We thank David Harlan, Marvin Gershengorn, and Derek LeRoith for their comments on the manuscript and Arnold Kriegstein for helpful discussions. We are grateful to Ron McKay for the gift of anti-nestin antibody, Chris Wright and Joel Habener for the gifts of anti-PDX-1 antibodies, and Bill Pavan for anti-Sox10 antibody. We thank Oksana Gavrilova and Stephanie Pack for their help with IEF isolations, Carolyn Smith for her help with laser scanning confocal microscopy, and Cheol Hong Park and Kristina Buac for their help with cell culture and preparation of the Figures.
A work supporting our finding of neural differentiation capacity of adult mouse pancreas (Seaberg, R.M. et al. Clonal Identification of Multipotent Precursors from Adult Mouse Pancreas that Generate Neural and Pancreatic Lineages. Nature Biotechnology 22, 1115–1124, 2004) had appeared in print after our manuscript was accepted for pulication.
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