Cellular progenitors remain poorly characterized in many adult tissues, limited in part by the lack of unbiased techniques to identify progenitors and their progeny. To address this fundamental problem, we developed a novel DNA analog-based lineage-tracing technique to detect multiple rounds of cell division in vivo. Here, we apply this technique to determine the adult lineage mechanism of the insulin-secreting beta cells of pancreatic islets, an important unresolved question in diabetes research. As expected, gastrointestinal and skin epithelia involve specialized progenitors that repeatedly divide to give rise to postmitotic cells. In contrast, specialized progenitors do not contribute to adult beta cells, not even during acute beta cell regeneration. Instead, beta cells are the products of uniform self-renewal, slowed by a replication refractory period that prevents beta cells from immediately redividing. Our approach provides unbiased resolution of previously inaccessible developmental niches and can elucidate lineage mechanisms without candidate markers.

Although many signaling pathways have been shown to promote ␤-cell growth, surprisingly little is known about the normal life cycle of preexisting ␤-cells or the signaling pathways required for ␤-cell survival. Adult ␤-cells have been speculated to have a finite life span, with ongoing adult ␤-cell replication throughout life to replace lost cells. However, little solid evidence supports this idea. To more accurately measure adult ␤-cell turnover, we performed continuous long-term labeling of proliferating cells with the DNA precursor analog 5-bromo-2-deoxyuridine (BrdU) in 1-year-old mice. We show that ␤-cells of aged adult mice have extremely low rates of replication, with minimal evidence of turnover. Although some pancreatic components acquired BrdU label in a linear fashion, only 1 in ϳ1,400 adult ␤-cells were found to undergo replication per day. We conclude that adult ␤-cells are very long lived. Diabetes 54: 2557-2567, 2005 I nadequate islet mass is a central finding in both type 1 and type 2 diabetes, resulting in an absolute or relative insulin deficiency and subsequent metabolic complications (1-3). Over the past decade, much has been learned about the specific signaling pathways that direct embryonic islet development (4) and postnatal islet growth (5). To compensate for increased metabolic demands, adult islet mass increases dramatically over the first year of life in rodents (6) and may represent growth within preexisting islets because islet density does not typically increase as mice age (7,8). It has been known for some time that adult ␤-cell growth is dependent on cyclindependent kinase-4 (9). Recently, we (10) and others (11) showed that cyclins D2 and D1 are the likely partners with cyclin-dependent kinase-4 to promote G 1 cell cycle progression in adult ␤-cells. Remarkably, new evidence suggests that much of adult ␤-cell growth may occur by replication of insulin-positive cells and not by ␤-cell neogenesis (12). However, it is possible that ␤-cell neogenesis may exist under some circumstances (13,14). Surprisingly little is known about the factors that promote adult ␤-cell survival, although some amount of ␤-cell apoptosis occurs in normal islets (15) as well as in pathological states (16). Although the absolute ␤-cell death rate is unknown, ␤-cell death appears to be a rare event. Supporting this notion, ␤-cell area increases severalfold throughout adulthood, despite an apparent decline in daily proliferation rate (6,17).Despite the lack of direct evidence, it has been largely assumed that ␤-cells have a finite life span, with dying cells replenished by new ␤-cells on an ongoing basis (12,14,17). One method of estimating cell life span is to measure the growth of total cell populations and cellular proliferation rate. In this model, the cell turnover rate is reflected by the proportion of cellular proliferation that does not contribute to cell population growth. In the simplest example of a stable population of cells with no net growth, cellular proliferation must be explained by ongoing lo...

Stem cells are proposed to segregate chromosomes asymmetrically during self-renewing divisions so that older ('immortal') DNA strands are retained in daughter stem cells whereas newly synthesized strands segregate to differentiating cells. Stem cells are also proposed to retain DNA labels, such as 5-bromo-2-deoxyuridine (BrdU), either because they segregate chromosomes asymmetrically or because they divide slowly. However, the purity of stem cells among BrdU-label-retaining cells has not been documented in any tissue, and the 'immortal strand hypothesis' has not been tested in a system with definitive stem cell markers. Here we tested these hypotheses in haematopoietic stem cells (HSCs), which can be highly purified using well characterized markers. We administered BrdU to newborn mice, mice treated with cyclophosphamide and granulocyte colony-stimulating factor, and normal adult mice for 4 to 10 days, followed by 70 days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of all BrdU-retaining haematopoietic cells were HSCs, revealing that BrdU has poor specificity and poor sensitivity as an HSC marker. Sequential administration of 5-chloro-2-deoxyuridine and 5-iodo-2-deoxyuridine indicated that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of the label. Thus, HSCs cannot be identified on the basis of BrdU-label retention and do not retain older DNA strands during division, indicating that these are not general properties of stem cells.