No Pancreatic Endocrine Stem Cells?
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《新英格兰医药杂志》
Advances in clinical islet transplantation have raised the hope that a definitive treatment for diabetes is close. However, widespread application of islet transplantation has been limited by a shortage of donor pancreases, and emphasis has therefore been placed on devising techniques to generate insulin-producing beta cells in vitro before their transplantation into patients. A large commitment of resources from the National Institutes of Health and private foundations is directed at bringing that hope to fruition. Where, then, should we start in our effort to generate more beta cells? The two principal candidates are embryonic stem cells and various types of adult cells. A recent article by Dor and coworkers1 questions the existence of adult pancreatic endocrine stem cells, which, by definition, are not yet beta cells but are often suggested as a potential source of beta cells for patients with diabetes.
The regeneration of beta cells after injury has been thought to involve the differentiation of stem cells that appear to reside within the pancreatic-duct epithelium. That belief is based on immunohistochemical evidence of large numbers of single beta cells arising in and around the ducts after pancreatic injury. Rather than relying on a histologic approach, Dor and colleagues worked out a way to label, through a specific exogenous stimulus, mature beta cells and their progeny and thereby track their fate during turnover and regeneration.
The authors engineered a transgenic mouse harboring two tweaked genes. One encoded a hybrid protein — part recombinase (an enzyme that rearranges DNA) and part estrogen receptor. This hybrid gene was hitched to the insulin promoter in order to restrict the expression of the hybrid gene to beta cells. The other gene encoded human placental alkaline phosphatase (HPAP), which in the presence of an appropriate substrate, results in a blue color at sites where it is expressed. The HPAP gene was modified so that it was expressed only on activation by the recombinase (Figure 1). To complete the signaling, tamoxifen was placed in the culture medium in order to bind the estrogen-receptor part of the hybrid protein, prompting it to move into the nucleus and activate the HPAP gene. By giving the mice pulsed doses of tamoxifen (thus labeling their beta cells) and then killing them at staggered intervals, before or after a partial pancreatectomy, the authors were able to obtain a series of snapshots of beta cells during normal turnover and regeneration. By looking at the snapshots, they were able to determine whether newly formed beta cells arose from preexisting beta cells (because they were positive for the heritable marker) or from other cells (because they were negative for the marker) (Figure 1).
Figure 1. Tracking the Beta Cell.
A recent study of the source of beta cells used an elegant strategy to irreversibly tag beta cells and, ultimately, their progeny in existing islets.1 The authors engineered a transgenic mouse to express a tamoxifen-inducible form of DNA recombinase in beta cells by placing the enzyme under the control of the insulin gene promoter. A pulse of tamoxifen was used to label existing beta cells. The fate of the labeled cells and of their progeny was followed after regeneration and normal islet turnover. In both settings, the new islets were composed of labeled beta cells, and the levels of these cells were identical to those of beta cells at the time of the tamoxifen pulse. This result indicates that new beta cells arose through the replication of existing beta cells. Had the new beta cells arisen from other cells (the putative stem cells), one would have expected to see a decreased level of labeled beta cells in the islets.
Their first experiment confirmed the widely held view that normal turnover of beta cells occurs through the replication of preexisting beta cells. The second and much more intriguing result was that after partial pancreatectomy, newly formed beta cells also arose from preexisting beta cells. Does this mean that there is no such thing as a pancreatic endocrine stem cell? The answer is "probably," since new beta cells could have been labeled after the pulsed dose of tamoxifen had been administered. A potential limitation of this approach is that the engineered recombinase can retain some activity in the absence of tamoxifen when expressed from strong promoters and over prolonged periods.2
The new results must be reconciled with the observation that many single beta cells appear in and near pancreatic ducts after injury.3 Where do these cells originate? According to the model suggested by the experiments of Dor and colleagues, they must come from preexisting beta cells. Since rodents, unlike humans, have very few isolated endocrine cells outside of islets, one explanation is that beta cells migrate from residual islets to the periductular space and beyond to coalesce with other endocrine cells and form new islets. An alternative scenario is that rare extraislet endocrine cells in the rodent undergo massive proliferation to form new islets, but this possibility is inconsistent with the findings of Dor et al.
The new study places increased emphasis on promoting the replication of adult beta cells4 and the differentiation of beta cells from embryonic stem cells at the expense of pursuing research into the use of adult stem cells for beta-cell replacement. With respect to the replication of beta cells, the challenges are substantial: human beta cells replicate less readily than those of mice, and replicating human beta cells undergo dedifferentiation and senescence in vitro.5 Last, but by no means least, are the ramifications of the study for federally mandated guidelines that restrict research on embryonic stem cells. This study will probably increase calls to lift those restrictions.
Dr. Levine reports holding equity in PanCell.
Source Information
From the Burnham Institute and the University of California, San Diego.
References
Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004;429:41-46.
Guo C, Yang W, Lobe CG. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 2002;32:8-18.
Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE. A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development. Diabetes 1993;42:1715-1720.
Hayek A, Beattie GM, Cirulli V, Lopez AD, Ricordi C, Rubin JS. Growth factor/matrix-induced proliferation of human adult beta-cells. Diabetes 1995;44:1458-1460.
Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F. Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro. J Endocrinol 2000;166:103-109.(Fred Levine, M.D., Ph.D.,)
The regeneration of beta cells after injury has been thought to involve the differentiation of stem cells that appear to reside within the pancreatic-duct epithelium. That belief is based on immunohistochemical evidence of large numbers of single beta cells arising in and around the ducts after pancreatic injury. Rather than relying on a histologic approach, Dor and colleagues worked out a way to label, through a specific exogenous stimulus, mature beta cells and their progeny and thereby track their fate during turnover and regeneration.
The authors engineered a transgenic mouse harboring two tweaked genes. One encoded a hybrid protein — part recombinase (an enzyme that rearranges DNA) and part estrogen receptor. This hybrid gene was hitched to the insulin promoter in order to restrict the expression of the hybrid gene to beta cells. The other gene encoded human placental alkaline phosphatase (HPAP), which in the presence of an appropriate substrate, results in a blue color at sites where it is expressed. The HPAP gene was modified so that it was expressed only on activation by the recombinase (Figure 1). To complete the signaling, tamoxifen was placed in the culture medium in order to bind the estrogen-receptor part of the hybrid protein, prompting it to move into the nucleus and activate the HPAP gene. By giving the mice pulsed doses of tamoxifen (thus labeling their beta cells) and then killing them at staggered intervals, before or after a partial pancreatectomy, the authors were able to obtain a series of snapshots of beta cells during normal turnover and regeneration. By looking at the snapshots, they were able to determine whether newly formed beta cells arose from preexisting beta cells (because they were positive for the heritable marker) or from other cells (because they were negative for the marker) (Figure 1).
Figure 1. Tracking the Beta Cell.
A recent study of the source of beta cells used an elegant strategy to irreversibly tag beta cells and, ultimately, their progeny in existing islets.1 The authors engineered a transgenic mouse to express a tamoxifen-inducible form of DNA recombinase in beta cells by placing the enzyme under the control of the insulin gene promoter. A pulse of tamoxifen was used to label existing beta cells. The fate of the labeled cells and of their progeny was followed after regeneration and normal islet turnover. In both settings, the new islets were composed of labeled beta cells, and the levels of these cells were identical to those of beta cells at the time of the tamoxifen pulse. This result indicates that new beta cells arose through the replication of existing beta cells. Had the new beta cells arisen from other cells (the putative stem cells), one would have expected to see a decreased level of labeled beta cells in the islets.
Their first experiment confirmed the widely held view that normal turnover of beta cells occurs through the replication of preexisting beta cells. The second and much more intriguing result was that after partial pancreatectomy, newly formed beta cells also arose from preexisting beta cells. Does this mean that there is no such thing as a pancreatic endocrine stem cell? The answer is "probably," since new beta cells could have been labeled after the pulsed dose of tamoxifen had been administered. A potential limitation of this approach is that the engineered recombinase can retain some activity in the absence of tamoxifen when expressed from strong promoters and over prolonged periods.2
The new results must be reconciled with the observation that many single beta cells appear in and near pancreatic ducts after injury.3 Where do these cells originate? According to the model suggested by the experiments of Dor and colleagues, they must come from preexisting beta cells. Since rodents, unlike humans, have very few isolated endocrine cells outside of islets, one explanation is that beta cells migrate from residual islets to the periductular space and beyond to coalesce with other endocrine cells and form new islets. An alternative scenario is that rare extraislet endocrine cells in the rodent undergo massive proliferation to form new islets, but this possibility is inconsistent with the findings of Dor et al.
The new study places increased emphasis on promoting the replication of adult beta cells4 and the differentiation of beta cells from embryonic stem cells at the expense of pursuing research into the use of adult stem cells for beta-cell replacement. With respect to the replication of beta cells, the challenges are substantial: human beta cells replicate less readily than those of mice, and replicating human beta cells undergo dedifferentiation and senescence in vitro.5 Last, but by no means least, are the ramifications of the study for federally mandated guidelines that restrict research on embryonic stem cells. This study will probably increase calls to lift those restrictions.
Dr. Levine reports holding equity in PanCell.
Source Information
From the Burnham Institute and the University of California, San Diego.
References
Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004;429:41-46.
Guo C, Yang W, Lobe CG. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 2002;32:8-18.
Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE. A second pathway for regeneration of adult exocrine and endocrine pancreas: a possible recapitulation of embryonic development. Diabetes 1993;42:1715-1720.
Hayek A, Beattie GM, Cirulli V, Lopez AD, Ricordi C, Rubin JS. Growth factor/matrix-induced proliferation of human adult beta-cells. Diabetes 1995;44:1458-1460.
Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F. Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro. J Endocrinol 2000;166:103-109.(Fred Levine, M.D., Ph.D.,)