MINIREVIEW
Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus

Andreas Lechner and Joel F. Habener

Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02114


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

In view of the recent success in pancreatic islet transplantation, interest in treating diabetes by the delivery of insulin-producing beta -cells has been renewed. Because differentiated pancreatic beta -cells cannot be expanded significantly in vitro, beta -cell stem or progenitor cells are seen as a potential source for the preparation of transplantable insulin-producing tissue. In addition to embryonic stem (ES) cells, several potential adult islet/beta -cell progenitors, derived from pancreas, liver, and bone marrow, are being studied. To date, none of the candidate cells has been fully characterized or is clinically applicable, but pancreatic physiology makes the existence of one or more types of adult islet stem cells very likely. It also seems possible that pluripotential stem cells, derived from the bone marrow, contribute to adult islet neogenesis. In future studies, more stringent criteria should be met to clonally define adult islet/beta -cell progenitor cells. If this can be achieved, the utilization of these cells for the generation of insulin-producing beta -cells in vitro seems to be feasible in the near future.

pancreatic islets; liver oval cells; embryonic stem cells; nestin; insulin; islet-like clusters; beta -cell; transplantation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

THE PREVALENCE OF THE DISABLING DISEASE diabetes mellitus is increasing at epidemic proportions throughout the populations of the world. It is estimated that 100 million individuals currently suffer from diabetes, 16 million in the United States alone. Diabetes comes about by the progressive failure of the beta -cells of the endocrine pancreas (islets of Langerhans) to produce the hormone insulin in the amounts required to meet the body's needs to maintain nutrient homeostasis. A lack of insulin results in elevations in blood glucose levels (hyperglycemia) and the subsequent development of premature cardiovascular disease, stroke, and kidney failure. There is no currently available permanent cure for diabetes. Blood glucose levels can be somewhat controlled by daily insulin shots or, in moderate cases of diabetes, by oral hypoglycemic (blood glucose-lowering) drugs. Diabetes is manifested in two relatively distinct forms: type 1 juvenile and type 2 adult onset. Type 1 juvenile diabetes is due to a nearly complete destruction of the beta -cells by processes of autoimmunity in which the body's immune system mistakenly attacks and destroys the beta -cells. The causation of type 2 adult-onset diabetes is more complex and poorly understood, but the beta -cells fail to produce adequate amounts of insulin in the face of the accompanying resistance of peripheral tissues to the actions of insulin. All patients with type 1 diabetes require daily insulin shots to survive. Twenty to thirty percent of type 2 diabetic individuals also require exogeneous insulin to control their hyperglycemia after oral hypoglycemic agents have failed.

Recently, hope for a permanent cure of diabetes has appeared, namely, the transplantation of islets isolated from donor pancreata into the livers of diabetic patients. The recent success of the Edmonton Protocol for pancreatic islet transplantation (45, 46) has sparked new interest in transplantation of insulin-producing cells. However, the amount of donor islet tissue is severely limited and will allow for the treatment of only a small fraction of patients with insulin-dependent diabetes, <0.5% of needy recipients. Moreover, differentiated beta -cells cannot be expanded efficiently in vitro (24). Therefore, multiple approaches are now being explored to generate insulin-producing cells in vitro, either by genetic engineering of beta -cells or by utilizing various potential beta -cell precursor cells, stem/progenitor cells, with the ability to grow in vitro and to differentiate into beta -cells. Some promising results have already been obtained with embryonic stem cells (ES cells) of both rodent and human origin (2, 38, 50). However, the potential use of ES cells for the treatment of diseases in humans is beclouded in controversy because of the ethical issues.

This review will focus on the potential of adult tissue-derived stem cells, in lieu of embryo-derived stem cells, for the treatment of diabetes. We discuss the role of adult islet stem/progenitor cells in normal physiology, highlight possible candidate cells isolated to date, and describe different approaches for stem cell-based therapy. We will also propose several criteria for the establishment of beta -cell differentiation from putative stem cells, both in vitro and in vivo.


    PHYSIOLOGICAL ROLE OF ADULT ISLET PROGENITOR CELLS
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

Stem or progenitor cells are defined by their capacity for self-renewal and asymmetric cell division, which leads to their differentiation and the formation of one or more mature tissues and the preservation of the stem cell population. Pluripotential cells exist in the inner mass of the blastocyst, so-called ES cells, and in the developing gonadal ridge, germ stem cells or GSCs. In some tissues, like bone marrow or intestinal epithelium, the existence of adult stem cells has been recognized for a long time and has been studied extensively (see review in Ref. 4). For other adult tissues, like brain, muscle, liver, and the endocrine pancreas, the function of adult stem cells is less well established, although substantial evidence for their existence and potential efficacy has recently been obtained.

When the embryonic development of the pancreas is studied, several sequentially expressed transcription factors are used to identify different cell populations within the forming pancreas that buds from the endodermal epithelium of the developing foregut. The homeodomain transcription factor Pdx-1 (also known as Ipf-1, Idx-1, and Stf-1) is expressed early in pancreas development (embryonic day 8.5 in the mouse), and its expression is required for the pancreas to develop. The disruption of Pdx-1 expression in mice and humans results in pancreatic agenesis, a complete failure of the pancreas to develop (31, 52). The expression of neurogenin 3 (Ngn3), a basic helix-loop-helix transcription factor, at embryonic development day 9.5 defines the first identifiable endocrine cell precursors. From then on, a cascade of transcription factors leads to the formation of all endocrine lineages of the islets of Langerhans (see reviews in Refs. 11 and 54). Throughout fetal development, a massive expansion of pancreatic endocrine cells is achieved mainly by the proliferation and subsequent differentiation of progenitor cells, rather than by the division of fully differentiated endocrine cells (8, 32, 42).

The formation of new islet tissue via the differentiation of stem/progenitor cells in adult pancreata was first demonstrated in different models of pancreatic injury. This regenerative process is called neogenesis. For example, partial pancreatectomy in rodents leads to the neogenesis of new islets from cells residing within or adjacent to the epithelium of pancreatic ducts (5). In another model of injury, the treatment of rodents with the drug streptozotocin (STZ), a beta -cell toxin, induces the regeneration of endocrine cells from intraislet progenitors (17, 21). Also, in the setting of a sudden increase in insulin demand, such as an experimental glucose infusion, neogenesis of endocrine cells from adult progenitor cells is a major adaptive mechanism (3). It was also shown recently that Ngn3-expressing cells persist into postnatal life and continue to contribute to islet cell development under normal physiological conditions (19). However, we suggest that the Ngn3-expressing cells are probably only transitory cells in endocrine maturation and not true stem or progenitor cells capable of self-renewal. Therefore, although the existence of adult islet progenitor cells can be inferred from these studies, the exact nature and location of these cells have yet to be definitively determined. It seems likely that distinct sets of progenitor cells and pathways of neogenesis (e.g., intraislet vs. duct to islet) exist in parallel within the pancreas. In this regard, it has been shown that the islets are polyclonal in origin (10).

A further level of complexity is added by the recent reports about the possible existence of circulating pluripotential and mobile stem cells that may take up residence within many different adult organs and tissues (4, 29). Although the uptake of circulating stem cells by the endocrine pancreas has not yet been demonstrated, the regeneration of endocrine cells from a systemic source of pluripotential stem cells seems possible.


    CANDIDATES FOR ADULT PANCREATIC STEM/PROGENITOR CELLS
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

To date, no adult pancreatic stem cell has been fully characterized. However, several candidate cells have been identified, isolated, and partially characterized (Table 1). It is important to note that differentiation protocols at present allow for only small amounts of insulin production compared with pancreatic islets.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Summary of studies of stem/progenitor cells and diabetes

Pancreatic Duct-Derived Stem/Progenitor Cells

Because under certain conditions islet regeneration appears to originate from the ductal epithelium (5), ductal tissue provides promising starting material for the search for islet progenitor cells. Indeed, the first report to describe in vitro-generated insulin-producing islet-like clusters (ILCs) was based on the expansion of cells from a crude preparation of mouse pancreatic ducts (44). The authors manually isolated ducts from collagenase-digested pancreata of prediabetic nonobese diabetic (NOD) mice. Rapidly growing cells could be maintained in culture for >3 yr and aggregated into clusters (ILCs), which could secrete small amounts of insulin in vitro. Upon transplantation into NOD mice, these ILCs significantly lowered the plasma glucose levels of the animals. However, the specific cells in the pancreatic ducts that are the progenitors giving rise to the insulin-producing cells were not identified or characterized.

Bonner-Weir et al. (7) generated ILCs in vitro from fractions of digested human pancreata enriched for ductal tissue. When plastic adherent cells from these preparations were overlaid with Matrigel, they formed cysts and clusters (cultured human islet buds, or CHIBs). Most cells in these aggregates were positive for the ductal marker cytokeratin 19, and others showed immunoreactivity for insulin and other islet hormones. The insulin content of the cultures increased over time, and a low level of glucose-responsive insulin secretion was observed in vitro. However, the capacity to expand the cultivated tissue was limited.

Nestin-Positive Islet-Derived Progenitor Cells

Our laboratory has reported that cells expressing the intermediate filament protein nestin, a marker of neural stem cells, can be isolated from human and rodent islets and expanded extensively in vitro. Insulin, glucagon, and Pdx-1/Ipf-1 expression, as well as low-level insulin secretion, can be detected in cultures of nestin-positive islet-derived stem/progenitor cells (NIPs) after addition of differentiating cytokines and growth factors (1, 64). Furthermore, the expression of other neuroendocrine, hepatic, and pancreatic exocrine genes can be demonstrated by RT-PCR (64). These cells also form ILCs in vitro, a process that is markedly enhanced by the addition of the insulinotropic, neogenic hormone glucagon-like peptide-1 (GLP-1) (1).

Whereas nestin was initially believed to be a marker restricted to neural and muscular progenitor cells (63), it is now recognized that certain mesenchymal cells also express nestin under selected conditions. With regard to the pancreas, recent reports demonstrate the expression of nestin in embryonic mesenchyme (48) and adult pancreatic stellate cells, as well as in proliferating vascular endothelium (36). Data from our own laboratory confirm these observations. Thus nestin in the pancreas is not an exclusive marker for islet stem/progenitor cells, but rather, some nestin-positive cells have the potential for the differentiation into pancreatic (islet) endocrine cells, including cells that produce insulin. This finding is also supported by the observation that nestin expression occurs as an intermediate step in the differentiation of beta -cells from ES cells (38). We also demonstrated recently, by flow cytometry cell sorting, that 1-3% of NIPs have a side-population phenotype similar to undifferentiated bone marrow stem cells (18, 37). This finding suggests that NIPs might contain a subpopulation of immature stem cells with a differentiating potential that extends beyond the endocrine pancreas, possibly as part of a population of adult multipotential/pluripotential stem cells.

Hepatic Oval Cells

The close anatomic association of pancreas and liver development from the primitive foregut during embryogenesis has prompted attempts to isolate pancreatic progenitor cells from adult liver. Recently, Yang et al. (60) reported the in vitro generation of ILCs from rat liver preparations enriched for hepatic oval cells. Oval cells are considered to be hepatic stem cells that can give rise to hepatocytes and bile duct cells (41). The authors induced in vivo proliferation of oval cells by a chemical injury of the liver and then enriched them to over 95% by flow cytometry cell sorting. After the in vitro expansion of the oval cells and their formation into ILCs, the expression of endocrine hormones and several other beta -cell markers could be induced, including low levels of insulin secretion. In preliminary in vivo studies, the authors report the successful reversal of diabetes in one STZ-treated NOD-scid mouse (60).

Multipotent Adult Progenitor Cells

Recently, the laboratory of Catherine Verfaillie (Jiang et al., Ref. 29) reported the successful expansion of cells from the adult bone marrow that have the potential to differentiate into ectodermal (neuronal), mesodermal (vascular endothelium), and endodermal (liver) cell types in vitro. In vivo, these cells show similar plasticity and contribute to multiple tissues after transplantation (29). The multipotent adult progenitor cells (MAPCs) can also be isolated from brain and muscle tissue (30) and may exist in every tissue of the body. The differentiation of MAPCs into pancreatic endocrine cells has not yet been shown. If we consider, however, that MAPCs can be transdifferentiated into hepatocyte-like cells with remarkable functional properties in vitro (47), the generation of pancreatic endocrine cells from MAPCs seems to be within reach.


    STEM CELL STIMULATORS AND DIFFERENTIATORS
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

Multipotential stem/progenitor cells can be induced to proliferate and to differentiate when exposed in vitro to certain growth factors or cytokines. A cell line (AR42J) derived from pancreatic ducts can be converted into insulin-producing cells by exposure to the growth factors betacellulin and activin A (39) or to the hepatic growth factor (40). Cells derived from pancreatic islets increase their rates of proliferation in the presence of basic fibroblast and epidermal growth factors (bFGF and EGF) (64). These cells can then be differentiated into ILCs by the removal of bFGF and EGF and the addition of differentiating factors such as activin A, betacellulin, hepatic growth factor (64), or the insulinotropic neogenic hormone GLP-1 (1). Nicotinamide is often used to increase the number of beta -cells and to boost the production of insulin (44). GLP-1 appears to be a particularly effective agent for inducing the differentiation of both NIPs (1) and duct cells into insulin-producing cells. Notably, GLP-1 induces the expression of the homeodomain protein Pdx-1/Ipf-1 in the stem/progenitor (1) and duct cells (26, 51, 59, 62), which appears to be essential for their differentiation into beta -cells. GLP-1 receptors have been identified on NIPs (1). It is believed that the activation of the receptor by GLP-1 activates Pdx-1 both by its phosphorylation via MAP kinase pathways and by effecting its translocation into the nucleus (9, 13, 27, 57). In many respects, Pdx-1 is a "master regulator" of pancreas development and function (see reviews in Refs. 11, 22, and 23). Pdx-1 is required for the pancreas to develop and is a key regulator of the expression of many beta -cell genes, including the insulin gene. The forced expression of Pdx-1 in a pancreatic ductal cell line renders the cells responsive to GLP-1, resulting in their differentiation into insulin-producing cells (26). Remarkably, the infection of a subpopulation of liver cells with an adenoviral vector expressing Pdx-1 converts the cells into functional beta -cells and restores glycemic control in mice rendered diabetic by their treatment with STZ (15). These studies suggest that, in the milieu of a stem cell, Pdx-1 in and by itself may be capable of programming the genome of the stem cell to express the set of genes required to establish a fully functional beta -cell.


    POSSIBLE ORIGINS OF PANCREAS AND OTHER ADULT ORGAN-DERIVED STEM CELLS
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

Although the existence of pluripotential or multipotential stem/progenitor cells in adult organs appears to be relatively well established, the intriguing question remains as to how these cells actually got there. First, they may represent a population of cells that are preserved throughout development in every single organ. These cells would have maintained many properties of pluripotential blastocyst cells and could participate in local tissue repair throughout the lifespan of the organism. A second possibility is that the new stem cells continuously reach different organs through the circulation; the bone marrow may contain a self-renewing population of multipotent stem cells that are continuously released into the circulation along with the different types of leukocytes. It is tempting to speculate that stem cells are an essential component of these circulating bone marrow surveillance cells and are prepared to home in on areas of injured tissues to participate in tissue repair and regeneration. One notion is that a normal physiological function of circulating stem cells is to replace differentiated cells that have a high turnover, such as intestinal mucosa, skin, and hair follicles (4). But their function may more importantly be to sense and home in on injured tissues. However, the naturally occurring population of circulating stem cells is likely to have a limited capacity to deal with severe injuries. This might be especially true with increasing age. This notion is consistent with the clinical concept of age-dependent loss of "organ reserve," the capacity to heal from injury and to replace injured tissue with functional new tissue.

There is considerable experimental evidence in support of the idea that circulating stem cells originating from the bone marrow populate adult organs (see review in Ref. 4). Patients transplanted with gender-mismatched hematopoietic stem cells (HSCs) developed a substantial degree of chimerism (up to 7%) in the liver, skin, and gastrointestinal tract, leading to the conclusion that circulating stem cells can differentiate into mature hepatocytes and epithelial cells of the skin and gastrointestinal tract (34). The mesenchymal MAPCs in the bone marrow recently described by Jiang et al. (30) have now been found in brain and muscle. Remarkably, gene expression profiling on oligonucleotide microarrays revealed that the genes expressed by MAPCs derived from brain, muscle, and bone marrow are 99.99% identical. These findings strongly indicate that the MAPCs found in these three different organs are the same cells and are consistent with the circumstance that MAPCs are produced in the bone marrow, delivered into the circulation, and taken up by brain and muscle. This mechanism for the distribution of stem cells is further supported by several studies in rodents with specific tissue injuries in which subpopulations of HSCs were administered systemically. In rodent models of heart attack (28, 33), brain stroke (25, 61), muscular dystrophy (20), and liver injury (35, 56), the cells rapidly home in on the sites of injuries and then differentiate into the resident tissue phenotype. The extent of repopulation of some of the injured tissues was substantial. In the mouse model of brain stroke, an average 34% of the vascular endothelial cells were derived from HSCs (25). Sixty percent or more of the liver tissue of some mice with liver injury consisted of hematopoietic donor cells (56).

The property of stem cells to colonize injured tissue is also demonstrated by the recently reported study by Quaini et al. (43). Female donor hearts transplanted into male recipients rapidly became chimeric; within 4-28 days after the transplant, 15, 12, and 9% of cells in myocytes, arterioles, and capillaries, respectively, of the donor heart consisted of recipient male gender cells. Although the origin of these recipient cells was uncertain, it was suggested that they likely came from circulating stem cells of the host recipient rather than from migration of cells into the heart from the recipient tissue adjacent to the anastomoses of the donor heart (43). It is noteworthy that the effective repair of the genetically determined mouse model of liver injury, due to an inborn error of tyrosine metabolism, has also been achieved by the administration of a population of pancreas-derived cells (55). Although the pancreatic cells that resulted in a repair of the liver were not characterized, it seems rather likely that the cells are related to the duct (7, 44) and/or are islet-derived (1, 64) stem/progenitor cells. The two models proposed for the origin of adult tissue-derived stem cells are not necessarily mutually exclusive. It seems reasonable to speculate that the pluripotent stem cells in the bone marrow are derived by lifetime self-renewal of the original pluripotent cells of the blastocyst and that tissue-specific multipotent progenitor cells with restricted differentiation capacity also exist.

The physiological and cellular mechanisms responsible for the "homing in" of circulating stem cells into areas of tissue injury is not understood. However, ligand-receptor interactions may be at play, as suggested by Jackson et al. (28), who conjectured that the vascular endothelial growth factor (VEGF) expressed in the injected HSCs may have directed them to the experimentally infarcted myocardium that expressed the VEGF receptor (Flk-1). In this regard, it is interesting to postulate that the expression of the GLP-1 receptor by pancreas-derived stem cells (NIPs) may promote homing in to the pancreatic islets, whose surface (mantle) is enriched in alpha -cells that express the proglucagon (GLP-1) gene. It seems possible to imagine that one function of the alpha -cells may be to "capture" circulating stem cells by virtue of their interaction with the GLP-1 hormone expressed by alpha -cells. If, indeed, the stem/progenitor cells in the pancreas originate from the bone marrow via the circulation, then it is possible that there may be separate distinct functions for the duct-derived compared with the islet-derived stem cells (Fig. 1). We envision that, once a pluripotential stem cell finds a niche in a particular tissue, it receives initial instructions from the environment to differentiate toward that particular tissue. Thereby, we suggest that the stem cells that are captured from the circulation by the ducts are instructed to become new islets, a concept consistent with the longstanding observations that new islets continue to bud off from the pancreatic ducts, the classic description of islet neogenesis. In contrast to the formation of new whole islets from the ducts, we conjecture that the stem cells that land in the islets are instructed to become new beta -cells to replace those that undergo natural senescence. This process of beta -cell neogenesis in fully formed islets may be different from the process of islet neogenesis that takes place in the ducts.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1.   Diagram depicting the hypothesis that pancreatic stem cells originate, at least in part, from circulating stem cells released from the bone marrow. It is conjectured that stem cells in the ducts are precursors of new islets (islet neogenesis), whereas stem cells within the islets are precursors of new beta -cells (beta -cell neogenesis). Islet neogenesis is a process that takes ~40 days to complete. beta -Cell neogenesis takes place 2-3 days after stimulation (3, 6, 12).


    ENVISIONED APPROACH FOR STEM CELL-DERIVED THERAPIES FOR DIABETES
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

At least two distinct approaches to the use of stem cells seem feasible for the treatment of diabetes (Fig. 2). One would be to procure appropriate donor tissue (pancreas biopsy) from a diabetic "to-be" recipient of islet transplants into the liver. The stem cells could be isolated from the biopsied tissue, expanded by culture in vitro, and differentiated into ILCs, or even into fully differentiated islets, once the technology to do so is available. Such a commercial source for "growing" unlimited numbers of islets would alleviate the acute shortage of donor pancreata from which islets for transplantation are prepared.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Envisioned stem cell approaches for the treatment of diabetes. Stem cells are isolated from a tissue biopsy, e.g., the pancreas. Stem cells are expanded in vitro and either differentiated into "islets" containing insulin-producing beta -cells and transplanted into the liver of diabetic patients or injected directly into the circulation, where they home in on the injured islets of insulin-dependent diabetic patients.

A second approach, quite different from the preparation of islets from stem cells in vitro, is to administer multipotential stem cells systemically to patients with type 1 diabetes and to depend on their properties to home in on injured tissues, as shown in the rodent studies. The cells would be delivered into the circulation as a "cellular medicine" by short infusions or injections. This approach would require the isolation and purification of a homogenous population of stem/progenitor cells that are selected for their high propensity to want to become islet or beta -cells. The coadministration during and after islet transplantation of stem cell stimulators/differentiators, such as GLP-1, would possibly enhance the efficiency of engraftment and the establishment of a permanent self-renewing population of stem/progenitor cells to provide a continued source of functional beta -cells.

Aspects of the proposed stem cell therapy that are important to recognize are the recent findings of the capacity of stem cells to induce tissue tolerance, in which the host recipient recognizes transplanted foreign tissue derived from stem cells as self, and the graft is not rejected. Such induction of tissue tolerance across both allogenic and xenogenic boundaries without a requirement for immunosuppression has been demonstrated in rodents in which mixed chimerism has been established by marrow transplants (see reviews in Refs. 53 and 58) and in rat and mouse models transplanted with stem cells (14, 49). It seems possible that stem cell transplants may also be able to resist the autoimmunity of type 1 diabetes, even after they differentiate into beta -cells in the recipient. Ferber et al. (16) recently reported the successful transdifferentiation of liver cells into fully functional beta -cells that achieve glycemic control in the NOD mouse model of autoimmunity type 1 diabetes.


    SUGGESTED CRITERIA TO DEFINE A PANCREAS-DERIVED STEM CELL
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

All studies of pancreatic endocrine progenitor cells published to date, including our own, fall short in fully defining their properties as stem/progenitor cells. To advance our understanding of islet biology and, more importantly, to isolate beta -cell stem/progenitors that will be clinically applicable, more rigorous methods are necessary. Therefore, we would like to suggest a set of criteria that should be met in future studies. 1) The stem or progenitor cell should be clonally isolated or marked; "enrichment" of a certain cell type alone is not sufficient. 2) In vitro differentiation to a fully functional beta -cell should be unequivocally established. Insulin expression per se does not make a particular cell a beta -cell. The expression of many other markers of beta -cells (e.g., Pdx1/Ipf1, GLUT2, and glucokinase) or other endocrine islet cells should be demonstrated. 3) Ultrastructural studies should confirm the formation of mature endocrine cells by identification of characteristic insulin secretory granules. 4) The in vitro function of endocrine cells, differentiated from stem cells, should be reminiscent of the natural counterparts. For beta -cells, this would imply a significant glucose-responsive insulin secretion, adequate responses to incretin hormones and secretagogues, and the expected electrophysiological properties. 5) In vivo studies in diabetic animals should demonstrate a reproducible and durable effect of the stem/progenitor-derived tissue on the attenuation of the diabetic phenotype. It should also be demonstrated that removal of the stem cell-derived graft after a certain period of time leads to reappearance of the diabetes. 6) For future clinical use, the tumorgenicity of stem/progenitor-derived tissue should be determined. Additionally, immune responses toward the transplanted cells should be examined.


    CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

To date, no fully defined and clinically applicable adult beta -cell stem/progenitor has been isolated. Nevertheless, studies of the development and the physiology of the pancreas make the existence of pancreatic stem/progenitor cells highly likely. Additionally, several potential candidate cells are being studied, and although more rigid experimental criteria have yet to be met, the published results look highly promising. The utilization of adult stem/progenitor cells for the generation of insulin-producing beta -cells in vitro and their use for the treatment of diabetes, therefore, seem to be feasible in the near future.


    ACKNOWLEDGEMENTS

We thank our colleagues in the Laboratory of Molecular Endocrinology for their support and helpful suggestions, Dr. Melissa K. Thomas for critical reading of the manuscript and suggestions, Robyn Blacken for expert experimental assistance, and Melissa Fannon for help in the preparation of the manuscript.


    FOOTNOTES

This study was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-30834, DK-55365, and DK-60125. A. Lechner is a research fellow supported by the Deutsche Forschungsgemeinschaft. J. F. Habener is an investigator with the Howard Hughes Medical Institute.

Address for reprint requests and other correspondence: J. F. Habener, Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit St., WEL 320, Boston, MA 02114 (E-mail: jhabener{at}partners.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00393.2002


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PHYSIOLOGICAL ROLE OF ADULT...
CANDIDATES FOR ADULT PANCREATIC...
STEM CELL STIMULATORS AND...
POSSIBLE ORIGINS OF PANCREAS...
ENVISIONED APPROACH FOR STEM...
SUGGESTED CRITERIA TO DEFINE...
CONCLUSIONS
REFERENCES

1.   Abraham, EJ, Leech CA, Lin JC, Zulewski H, and Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 143: 3152-3161, 2002[Abstract/Free Full Text].

2.   Assady, S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, and Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 50: 1691-1697, 2001[Abstract/Free Full Text].

3.   Bernard, C, Berthault MF, Saulnier C, and Ktorza A. Neogenesis vs. apoptosis as main components of pancreatic beta cell mass changes in glucose-infused normal and mildly diabetic adult rats. FASEB J 13: 1195-1205, 1999[Abstract/Free Full Text].

4.   Blau, HM, Brazelton TR, and Weimann JM. The evolving concept of a stem cell: entity or function? Cell 105: 829-841, 2001[ISI][Medline].

5.   Bonner-Weir, S, Baxter LA, Schuppin GT, and Smith FE. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42: 1715-1720, 1993[Abstract].

6.   Bonner-Weir, S, Deery D, Leahy JL, and Weir GC. Compensatory growth of pancreatic beta-cells in adult rats after short-term glucose infusion. Diabetes 38: 49-53, 1989[Abstract].

7.   Bonner-Weir, S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, and O'Neil JJ. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 97: 7999-8004, 2000[Abstract/Free Full Text].

8.   Bouwens, L, Lu WG, and De Krijger R. Proliferation and differentiation in the human fetal endocrine pancreas. Diabetologia 40: 398-404, 1997[ISI][Medline].

9.   Buteau, J, Roduit R, Susini S, and Prentki M. Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia 42: 856-864, 1999[ISI][Medline].

10.   Deltour, L, Leduque P, Paldi A, Ripoche MA, Dubois P, and Jami J. Polyclonal origin of pancreatic islets in aggregation mouse chimaeras. Development 112: 1115-1121, 1991[Abstract].

11.   Edlund, H. Pancreatic organogenesis---developmental mechanisms and implications for therapy. Nat Rev Genet 3: 524-532, 2002[ISI][Medline].

12.   Edvell, A, and Lindstrom P. Initiation of increased pancreatic islet growth in young normoglycemic mice (Umea +/?). Endocrinology 140: 778-783, 1999[Abstract/Free Full Text].

13.   Elrick, LJ, and Docherty K. Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1. Diabetes 50: 2244-2252, 2001[Abstract/Free Full Text].

14.   Fandrich, F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS, Parwaresch R, Zavazava N, and Binas B. Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat Med 8: 171-178, 2002[ISI][Medline].

15.   Ferber, S, Halkin A, Cohen H, Ber I, Einav Y, Goldberg I, Barshack I, Seijffers R, Kopolovic J, Kaiser N, and Karasik A. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia. Nat Med 6: 568-572, 2000[ISI][Medline].

16.  Ferber S, Perl S, Sapir T, Ohanuna Z, Benvenisti L, and Meivar-Levy I. Transconversion of liver into endocrine pancreas. In: Beta Cell Biology in the 21st Century. Bethesda, MD: Am. Physiol. Soc., November 26-28, 2001.

17.   Fernandes, A, King LC, Guz Y, Stein R, Wright CV, and Teitelman G. Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets. Endocrinology 138: 1750-1762, 1997[Abstract/Free Full Text].

18.   Goodell, MA, Brose K, Paradis G, Conner AS, and Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183: 1797-1806, 1996[Abstract].

19.   Gu, G, Dubauskaite J, and Melton DA. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors. Development 129: 2447-2457, 2002[Abstract/Free Full Text].

20.   Gussoni, E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, and Mulligan RC. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401: 390-394, 1999[ISI][Medline].

21.   Guz, Y, Nasir I, and Teitelman G. Regeneration of pancreatic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 142: 4956-4968, 2001[Abstract/Free Full Text].

22.   Habener, JF. Glucagonlike peptide-1 agonist stimulation of beta -cell growth and differentiation. Curr Opin Endocrinol Diabetes 8: 74-81, 2001.

23.   Habener, JF, and Stoffers DA. A newly discovered role of transcription factors involved in pancreas development and the pathogenesis of diabetes mellitus. Proc Assoc Am Physicians 110: 12-21, 1998[ISI][Medline].

24.   Halvorsen, TL, Beattie GM, Lopez AD, Hayek A, and Levine F. Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro. J Endocrinol 166: 103-109, 2000[Abstract/Free Full Text].

25.   Hess, DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, and Carothers J. Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke. Stroke 33: 1362-1368, 2002[Abstract/Free Full Text].

26.   Hui, H, Wright C, and Perfetti R. Glucagon-like peptide 1 induces differentiation of islet duodenal homeobox-1-positive pancreatic ductal cells into insulin-secreting cells. Diabetes 50: 785-796, 2001[Abstract/Free Full Text].

27.   Hussain, MA, and Habener JF. Glucagon-like peptide 1 increases glucose-dependent activity of the homeoprotein IDX-1 transactivating domain in pancreatic beta-cells. Biochem Biophys Res Commun 274: 616-619, 2000[ISI][Medline].

28.   Jackson, KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK, and Goodell MA. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107: 1395-1402, 2001[Abstract/Free Full Text].

29.   Jiang, Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, and Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418: 41-49, 2002[ISI][Medline].

30.   Jiang, Y, Vaessen B, Lenvik T, Blackstad M, Reyes M, and Verfaillie C. Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp Hematol 30: 896, 2002[ISI][Medline].

31.   Jonsson, J, Carlsson L, Edlund T, and Edlund H. Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371: 606-609, 1994[ISI][Medline].

32.   Kaung, HL. Growth dynamics of pancreatic islet cell populations during fetal and neonatal development of the rat. Dev Dyn 200: 163-175, 1994[ISI][Medline].

33.   Kocher, AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, and Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7: 430-436, 2001[ISI][Medline].

34.   Korbling, M, Katz RL, Khanna A, Ruifrok AC, Rondon G, Albitar M, Champlin RE, and Estrov Z. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 346: 738-746, 2002[Abstract/Free Full Text].

35.   Lagasse, E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, and Grompe M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6: 1229-1234, 2000[ISI][Medline].

36.   Lardon, J, Rooman I, and Bouwens L. Nestin expression in pancreatic stellate cells and angiogenic endothelial cells. Histochem Cell Biol 117: 535-540, 2002[ISI][Medline].

37.   Lechner, A, Leech CA, Abraham EJ, Nolan AL, and Habener JF. Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter. Biochem Biophys Res Commun 293: 670-674, 2002[ISI][Medline].

38.   Lumelsky, N, Blondel O, Laeng P, Velasco I, Ravin R, and McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292: 1389-1394, 2001[Abstract/Free Full Text].

39.   Mashima, H, Ohnishi H, Wakabayashi K, Mine T, Miyagawa J, Hanafusa T, Seno M, Yamada H, and Kojima I. Betacellulin and activin A coordinately convert amylase-secreting pancreatic AR42J cells into insulin-secreting cells. J Clin Invest 97: 1647-1654, 1996[Abstract/Free Full Text].

40.   Mashima, H, Shibata H, Mine T, and Kojima I. Formation of insulin-producing cells from pancreatic acinar AR42J cells by hepatocyte growth factor. Endocrinology 137: 3969-3976, 1996[Abstract].

41.   Petersen, BE, Goff JP, Greenberger JS, and Michalopoulos GK. Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27: 433-445, 1998[ISI][Medline].

42.   Polak, M, Bouchareb-Banaei L, Scharfmann R, and Czernichow P. Early pattern of differentiation in the human pancreas. Diabetes 49: 225-232, 2000[Abstract].

43.   Quaini, F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, Kajstura J, Leri A, and Anversa P. Chimerism of the transplanted heart. N Engl J Med 346: 5-15, 2002[Abstract/Free Full Text].

44.   Ramiya, VK, Maraist M, Arfors KE, Schatz DA, Peck AB, and Cornelius JG. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 6: 278-282, 2000[ISI][Medline].

45.   Ryan, EA, Lakey JR, Paty BW, Imes S, Korbutt GS, Bigam D, Rajotte RV, Kneteman NM, and Shapiro AM. Successful islet transplantation: continued insulin reserve provides long-term glycemic control. Diabetes 51: 2148-2157, 2002[Abstract/Free Full Text].

46.   Ryan, EA, Lakey JR, Rajotte RV, Korbutt GS, Kin T, Imes S, Rabinovitch A, Elliott JF, Bigam D, Kneteman NM, Warnock GL, Larsen I, and Shapiro AM. Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol. Diabetes 50: 710-719, 2001[Abstract/Free Full Text].

47.   Schwartz, RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu WS, and Verfaillie CM. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109: 1291-1302, 2002[Abstract/Free Full Text].

48.   Selander, L, and Edlund H. Nestin is expressed in mesenchymal and not epithelial cells of the developing mouse pancreas. Mech Dev 113: 189-192, 2002[Medline].

49.   Shizuru, JA, Weissman IL, Kernoff R, Masek M, and Sheffold YC. Purified hematopoietic stem cell grafts induce tolerance to alloantigens and can mediate positive and negative T cell selection. Proc Natl Acad Sci USA 97: 9555-9560, 2000[Abstract/Free Full Text].

50.   Soria, B, Roche E, Berna G, Leon-Quinto T, Reig JA, and Martin F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49: 157-162, 2000[Abstract].

51.   Stoffers, DA, Kieffer TJ, Hussain MA, Drucker DJ, Bonner-Weir S, Habener JF, and Egan JM. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 49: 741-748, 2000[Abstract].

52.   Stoffers, DA, Zinkin NT, Stanojevic V, Clarke WL, and Habener JF. Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat Genet 15: 106-110, 1997[ISI][Medline].

53.   Sykes, M. Mixed chimerism and transplant tolerance. Immunity 14: 417-424, 2001[ISI][Medline].

54.   Thomas, MK, and Habener JF. Pancreas development. In: Immunologically Mediated Endocrine Diseases, edited by Gill RG. Baltimore, MD: Lippincott Williams & Wilkins, 2002, p. 141-166.

55.   Wang, X, Al-Dhalimy M, Lagasse E, Finegold M, and Grompe M. Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells. Am J Pathol 158: 571-579, 2001[Abstract/Free Full Text].

56.   Wang, X, Montini E, Al-Dhalimy M, Lagasse E, Finegold M, and Grompe M. Kinetics of liver repopulation after bone marrow transplantation. Am J Pathol 161: 565-574, 2002[Abstract/Free Full Text].

57.   Wang, X, Zhou J, Doyle ME, and Egan JM. Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic beta-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism. Endocrinology 142: 1820-1827, 2001[Abstract/Free Full Text].

58.   Wekerle, T, and Sykes M. Mixed chimerism and transplantation tolerance. Annu Rev Med 52: 353-370, 2001[ISI][Medline].

59.   Xu, G, Stoffers DA, Habener JF, and Bonner-Weir S. Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats. Diabetes 48: 2270-2276, 1999[Abstract].

60.   Yang, L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, and Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA 99: 8078-8083, 2002[Abstract/Free Full Text].

61.   Zhang, ZG, Zhang L, Jiang Q, and Chopp M. Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 90: 284-248, 2002[Abstract/Free Full Text].

62.   Zhou, J, Wang X, Pineyro MA, and Egan JM. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 48: 2358-2366, 1999[Abstract].

63.   Zimmerman, L, Parr B, Lendahl U, Cunningham M, McKay R, Gavin B, Mann J, Vassileva G, and McMahon A. Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron 12: 11-24, 1994[ISI][Medline].

64.   Zulewski, H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, and Habener JF. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes 50: 521-533, 2001[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 284(2):E259-E266
0193-1849/03 $5.00 Copyright © 2003 the American Physiological Society