Mitogen Synergy: An Emerging Route to Boosting Human Beta Cell Proliferation

Abstract

Decreased number and function of beta cells are a key aspect of diabetes mellitus (diabetes), a disease that remains an onerous global health problem. Means of restoring beta cell mass are urgently being sought as a potential cure for diabetes. Several strategies, such as de novo beta cell derivation via pluripotent stem cell differentiation or mature somatic cell transdifferentiation, have yielded promising results. Beta cell expansion is another promising strategy, rendered challenging by the very low proliferative capacity of beta cells. Many effective mitogens have been identified in rodents, but the vast majority do not have similar mitogenic effects in human beta cells. Extensive research has led to the identification of several human beta cell mitogens, but their efficacy and specificity remain insufficient. An approach based on the simultaneous application of several mitogens has recently emerged and can yield human beta cell proliferation rates of up to 8%. Here, we discuss recent advances in restoration of the beta cell population, focusing on mitogen synergy, and the contribution of RNA-sequencing (RNA-seq) to accelerating the elucidation of signaling pathways in proliferating beta cells and the discovery of novel mitogens. Together, these approaches have taken beta cell research up a level, bringing us closer to a cure for diabetes.

Keywords: diabetes; endocrine beta cell; human vs. rodent; proliferation; signaling; synergy.

FIGURE 1

Principles of cell cycle in the beta cell. Beta cells predominantly reside in a quiescent, non-mitotic G0 phase. Upon the mitogenic stimuli, the assembly of Cyclins D and CDK4/6 complex and consequent pRb inactivation via hyperphosphorylation trigger the cell cycle entry. Once the E2F transcription factors are released from the pRb restraining, they enhance expression of the cell cycle promoting genes, including the late Cyclins and CDKs. The activities of different Cyclins-CDK complexes gradually replace each other with the cell cycle progression, so that each Cyclins-CDK complex is dominant during different cell cycle phases. In G1, Cyclins D-CDK4/6 regulate cell growth and preparation for DNA replication. In the S phase, Cyclins E-CDK2 complexes control DNA replication. Whereas in phase G2, Cyclins A with CDK1 are driving preparation for the cell division. Finally, in phase M, complexes of Cyclins B-CDK1 regulate the cell division. The promoting activities of Cyclins-CDK complexes are negatively regulated by the cell cycle inhibitors. Families of Ink4 and Cip/Kip inhibitors restrain the early and late Cyclins-CDKs complexes, respectively. On the way towards division, the cell must encounter several STOP checkpoints whereby the cell state, DNA integrity and the surrounding conditions are checked. Cells which do not pass the checkpoint, are blocked from cell cycle progression until the conditions improve or, in an irresolvable situation, undergo apoptosis. The inner circles on Figure 1 represent the commonly used proliferation markers: Ki67, BrdU, pHH3. Intensity of the violet color corresponds to the abundance of the marker at a given phase, with white color indicating absence of the marker and dark violet – the highest detectability.

FIGURE 2

Mitogenic synergy in human beta cell proliferation. The figure presents how application of mitogens alone or in combinations influences the beta cell proliferation. Several of the most potent mitogenic compounds are depicted in the top block, together with the key signaling events through which they regulate beta cell proliferation (the block below). The bottom block of the figure demonstrates how the values of beta cell proliferation rates increase upon the application of dual or triple mitogen combinations in comparison to the effects of the individual mitogens. Numbers in brackets indicate the literature references. *For all the mitogens, except LIF, the values of proliferation rates were obtained with the staining for Ki67 presence, and are therefore suitable for comparison. For LIF the proliferation rate has been obtained with the EdU incorporation assay. LIF is included into the scheme as the first mitogen described in a triple combination (LIF + Harmine + TGF-beta inhibitor).