Our experiments were designed to address the mechanism of growth and maintenance of mature β cells. To determine whether all β cells divide at the same rate in the adult mouse, two experiments were undertaken. The tetracycline-inducible H2BGFP and MADM systems are complementary approaches: whereas tetO-H2BGFP labels most β cells and provides a broad view of the population dynamic, MADM labels single cells and provides an accurate clonal analysis of the progeny of individual cells within the β cell pool. Both the uniform loss of the H2BGFP label with time in the β cell population and the comparable β cell clone sizes generated through MADM analysis indicate homogeneity exists within the β cell pool. Stated otherwise, all β cells appear to contribute equally to growth and maintenance.
The β cell mass is dynamic and can respond to environmental cues such insulin and glucose [25
]. The β cell number increases dramatically in the first year of rodent life [12
], up to 10-fold in cases of insulin resistance [27
], and up to 1.5-fold during pregnancy [28
]. Recent experiments suggest that when not hindered by persistent autoimmune attack or the toxicity of high blood glucose levels [30
], β cells have the capacity to regenerate. While the mechanism regulating β cell expansion remains unclear, our findings indicate that all β cells are capable of replication and are therefore viable targets for in vitro or in vivo expansion.
Seaberg et al. recently reported that single-cell clones derived from adult islets generated colonies of 2,000–10,000 cells that expressed markers of neural, glial, pancreatic endocrine, exocrine, and duct identities [31
]. These clones were generated from ~0.02% of islet cells, though their identity and relationship to in vivo growth is yet to be determined. We cannot rule out the possibility that a rare type of β cell was missed in our examination of individual clones using the MADM marking experiments. However, because the rate of clonal expansion is sufficient to account for the growth of the β cell population during the chase period, a rare highly proliferative β cell did not contribute significantly to the expansion of β cell mass.
Published rates for β cell replication in adult mice (12 wk old) are highly variable, from 2% [32
] to 15% per day [33
]. Assuming 5% of β cells replicate per day, and that all β cells are equivalent, β cells should divide approximately every 20 d. This would dilute the H2BGFP label beyond detection (by completing up to five rounds of replication) within 100 d. In addition, clone size at 2 mo should be approximately eight cells. These straightforward calculations predict results that are entirely consistent with our findings. These estimates, of course, assume no β cell death over the duration of our experiments.
β cells have a finite lifespan, but the absolute β cell death rate is unknown. Based on β cell mass measurements and an estimate of β cell proliferation of 2% per day throughout adulthood, Finegood et al. calculated the β cell lifespan to be 52 d [32
]. Recent findings demonstrate that β cell proliferation rates decline to less than 0.1% in 1-y-old mice [33
], casting doubt on the often quoted rates for β cell turnover in mice. Furthermore, TUNEL analysis of wild-type β cells consistently fail to identify apoptotic cells [13
]. Regardless of the true rate of β cell turnover, our findings of a uniform loss of label and a consistent clone size indicate that all β cells have equivalent replicative capacity.
Pancreatic β cells are not the only differentiated cell type capable of growth and maintenance without the support of an adult stem cell population. Hepatocytes are highly replicative and not thought to be supported by a facultative stem cell under normal conditions [35
]. Pulse–chase analysis with the tetracycline-inducible H2BGFP label shows that all hepatocytes lose their label at the same rate. Therefore, like the β cell population, the hepatocyte population seems to be homogeneous. We do not know of an example of a mature differentiated cell type that has two populations (one replicative and the other not). We speculate that when tissues are without an adult stem cell, they are replenished by equal replication of all differentiated cells.
The demonstration that all β cells are equivalent, contributing equally to the growth and maintenance of the β cell population, has clinical implications if we assume that rodents and man use the same mechanism for pancreatic homeostasis. The destruction of β cells that causes type I diabetes has been counteracted by the transplantation of β cells. The clinical impact of this approach is currently limited, in part, by the scarcity of available pancreatic tissue [36
]. A better understanding of adult β cell replication may help attempts to expand pancreatic β cells in vitro as a source of transplant material to treat diabetes.