Maturation of stem cell-derived beta-cells guided by the expression of urocortin 3

Abstract

Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts.

Figure 1. UCN3 expression as a late stage maturation marker for human alpha- and beta-cells

The figure provides a general overview of the (dis)appearance of notable markers that are expressed at key intermediate stages from early endocrine progenitor cells towards the alpha- and beta-cell lineage. UCN3 expression in beta-cells appears later than any of the other markers listed here, with the possible exception of the loss of MafB expression in mouse beta-cells. Note that the appearance of UCN3 in mature alpha-cells is unique for primate alpha-cells and does not occur in rodents islets.

Figure 2. Expression of UCN3 in mouse and human islets

In mouse islets, UCN3 (labeled in green) is expressed exclusively in beta-cells labeled in red for insulin (A). UCN3 is not expressed in mouse alpha-cells (arrows) or delta-cells (asterisks), labeled for glucagon in red and somatostatin in white, respectively (B). In contrast, UCN3 in human islets is expressed by both beta- and alpha-cells, as indicated by its co-localization with insulin (C) and glucagon (D), both in red. These panels were reprinted from [56] under the terms of the Creative Commons Attribution License (CCAL), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Figure 3. A beta-cell-centric view of the onset of expression of key genes involved in beta-cell development and maturation

Many of the genes required for normal beta-cell function are already expressed prior to the tertiary transition in which beta-cells mature and acquire glucose responsiveness. Initial expression for UCN3 [56, 77], ZNT8 [126], MAFA [73], Cx36 [127], PCSK1 and PCSK2 [128], MAFB [129], PAX4 [47], NEUROD [48], Islet1 [46], NGN3 [52], NKX6.1 [42], GLUT2 [130], and Pdx1 [58] is shown.

Figure 4. UCN3 expression follows the onset of MafA and MafB expression in mouse development

UCN3 expression overlaps with the expression of MafA (A) and MafB (B) at postnatal day 2 (indicated by arrows), when mouse beta-cells express both MafA and MafB. Note that MafB is also expressed in a number of UCN3-negative cells located at the periphery of the islet, indicated by asterisks. These cells are possibly alpha-cells, which retain MafB expression. In adult mouse islets, UCN3 and MafA expression in beta-cells continue to overlap (C, arrows), while MafB expression has been lost from beta-cells, and now labels only a UCN3-negative population of presumptive alpha-cells (D, asterisks). UCN3 was stained by a guinea pig anti-UCN3 antiserum (#44) developed in house. MafA and MafB were stained by rabbit anti-MafA and rabbit anti-MafB (both Bethyl Labs), using standard antigen retrieval, as described in detail in [56].

Figure 5. UCN3 expression follows the onset of insulin and glucagon expression in human pancreas development

At 55 days post coitus (dpc) numerous cells positive for insulin (red) and glucagon (white) can be readily observed, while there is only faint UCN3 immunoreactivity (A, green). Note the insulin/glucagon co-positive bi-hormonal cell in the circle. At 76 dpc (B) and 94 pdc (C), UCN3 is evidently expressed, and co-localizes with beta-cells (arrows) labeled for insulin (red) and with alpha-cells (asterisks) labeled for glucagon (white). While beta-cells, which do not yet co-express UCN3, can be observed at each of these three developmental stages, their frequency appears to go down as development progresses. Antisera and methods are as previously described in detail in [56]. Human fetal pancreas tissue sections were generously provided by Dr. Ruiyu Xie of the University of California, San Diego, who obtained them from the Birth Defects Research Laboratory of the University of Washington. The use of this material was certified as exempt from IRB approval by UCSD.