Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway

Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. DOI: http://dx.doi.org/10.7554/eLife.02809.00

Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy

The β-cell mitogenic effects of ANGPTL8 have been subjected to substantial debate. The original findings suggested that ANGPTL8 overexpression in mice induced a 17-fold increase in β-cell proliferation. Subsequent studies in mice contested this claim, but a more recent report in rats supported the original observations. These conflicting results might be explained by variable ANGPTL8 expression and differing methods of β-cell quantification. To resolve the controversy, three independent labs collaborated on a blinded study to test the effects of ANGPTL8 upon β-cell proliferation. Recombinant human betatrophin (hBT) fused to maltose binding protein (MBP) was delivered to mice by intravenous injection. The results demonstrate that ANGPTL8 does not stimulate significant β-cell proliferation. Each lab employed different methods for β-cell identification, resulting in variable quantification of β-cell proliferation and suggests a need for standardizing practices for β-cell quantification. We also observed a new action of ANGPTL8 in stimulating CD45+ hematopoietic-derived cell proliferation which may explain, in part, published discrepancies. Overall, the hypothesis that ANGPTL8 induces dramatic and specific β-cell proliferation can no longer be supported. However, while ANGPTL8 does not stimulate robust β-cell proliferation, the original experimental model using drug-induced (S961) insulin resistance was validated in subsequent studies, and thus still represents a robust system for studying signals that are either necessary or sufficient for β-cell expansion. As an added note, we would like to commend collaborative group efforts, with repetition of results and procedures in multiple laboratories, as an effective method to resolve discrepancies in the literature

Promotion of Reprogramming to Ground State Pluripotency by Signal Inhibition

Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency—including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras—were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1–2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon

Nanog Is the Gateway to the Pluripotent Ground State

SummaryPluripotency is generated naturally during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Pluripotency can be recreated by somatic cell reprogramming. Here we present evidence that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency. Production of pluripotent hybrids by cell fusion is promoted by and dependent on Nanog. In transcription factor-induced molecular reprogramming, Nanog is initially dispensable but becomes essential for dedifferentiated intermediates to transit to ground state pluripotency. In the embryo, Nanog specifically demarcates the nascent epiblast, coincident with the domain of X chromosome reprogramming. Without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state that is ultimately nonviable. These findings suggest that Nanog choreographs synthesis of the naive epiblast ground state in the embryo and that this function is recapitulated in the culmination of somatic cell reprogramming

ANGPTL8 treatment in mice increases non-β-cell proliferation.

<p><b>(a-b)</b> Staining performed by Lab #2 for insulin (yellow), EdU (green), Nkx6.1 (red), and DAPI (blue) for <b>(a)</b> MBP and <b>(b)</b> MBP+hBT islets. White arrowheads indicate a proliferating insulin⁺ Nkx6.1⁺ DAPI⁺ cell [inset <b>(a)</b>] and red arrowheads indicate a proliferating insulin^- Nkx6.1^- DAPI⁺ cell [inset <b>(b)</b>]. Scale bar: 100 μm. <b>(c-e)</b> Quantification of EdU⁺ proliferation for <b>(c)</b> β-cells, <b>(d)</b> total islet cells, and <b>(e)</b> non-β islet cells. <b>(f-h)</b> Quantification of <b>(f)</b> β-cell number, <b>(g)</b> total islet cell number, and <b>(h)</b> non-β islet cell number per islet. Islet cells were identified by dilating insulin area by one cell’s diameter and filling all holes within the object. β-cells were identified by Nkx6.1⁺ cells co-localized with DAPI surrounded by insulin. Non-β islet cells were calculated by subtracting the β-cell counts from the total islet cell counts. <b>(i)</b> Total islet cell proliferation by Ki67 from original stained slides by Lab #2 examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159276#pone.0159276.g003" target="_blank">Fig 3</a>. <b>(j-k)</b> Quantification of pancreatic proliferation by <b>(j)</b> Ki67⁺ or <b>(k)</b> EdU⁺ (% of total non-islet cells) from original stained slides by Lab #2 examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159276#pone.0159276.g003" target="_blank">Fig 3</a>. Data are mean ± SEM. MBP and MBP+hBT = 10 animals per group. Student’s <i>t</i> test was performed. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001.</p

EdU captures all proliferative events detected by Ki67.

<p><b>(a-b)</b> Immunostaining by Lab #2 for insulin (yellow), Ki67 (green), EdU (red), and DAPI (blue). Scale bar: 100 μm. Insets demonstrate <b>(a)</b> Ki67⁺ EdU⁺ co-positive cells and <b>(b)</b> a rare Ki67⁺ EdU- cell. <b>(c-d)</b> Quantification of Ki67⁺ cells co-expressing EdU in <b>(c)</b> all pancreatic cells and <b>(d)</b> β-cells. Data are reported as the mean ± SEM. MBP and MBP+hBT = 7 animals per group.</p

ANGPTL8 treatment in mice has no effect on β-cell proliferation.

<p><b>(a-d)</b> Staining for insulin (yellow) and <b>(a-b)</b> Ki67 (green) or <b>(c-d)</b> EdU (red) for <b>(a,c)</b> MBP and <b>(b,d)</b> MBP+hBT islets. Scale bar: 100 μm. <b>(e-j)</b> β-cell proliferation quantified by insulin⁺ DAPI⁺ cells containing <b>(e-g)</b> Ki67 or <b>(h-j)</b> EdU as a percentage of total β-cells, from two control groups (buffer injection alone or with EdU), MBP, and MBP+hBT samples. One-way ANOVA was performed with Bonferroni’s multiple comparison test. *** <i>p</i> < 0.001 MBP+hBT versus MBP, ** <i>p</i> < 0.01 MBP+hBT versus Buffer, no EdU and buffer, EdU. <b>(k-m)</b> Linear regression analysis of the correlation between EdU⁺ and Ki67⁺ β-cell proliferation. Data are mean ± SEM. Buffer, no EdU and buffer, EdU = 5 animals per group; MBP and MBP+hBT = 10 animals per group.</p

Highly proliferative cells in islets of ANGPTL8 treated mice are not glucagon, endocrine, epithelial, neuronal, myofibroblast, or vascular cells.

<p><b>(a)</b> Immunostaining for insulin (yellow), glucagon (green), EdU (red) and DAPI (blue). <b>(b)</b> Immunostaining for synaptophysin (green), EdU (red), and DAPI (blue). <b>(c-h)</b> Immunostaining for EdU (green) and DAPI (blue) with various makers (red); <b>(c)</b> E-cadherin, <b>(d)</b> N-cadherin, <b>(e)</b> smooth muscle actin α (SMAα), <b>(f)</b> desmin, <b>(g)</b> CD31, <b>(h)</b> CD34. Insets indicate EdU positive cells that do not express glucagon, synaptophysin, E-cadherin, N-cadherin, SMAα, desmin, CD31, or CD34. Scale bar = 100 μm.</p

Highly proliferative cells in islets of ANGPTL8 treated mice are not immune related cells.

<p>Immunostaining for EdU (green) and DAPI (blue), with various markers for immune-related cells (red) in <b>(a,c,e,g,i)</b> pancreatic islets or in <b>(b,d,f,h,j)</b> the spleen used as a positive control. <b>(a-b)</b> CD3 (T-cells), <b>(c-d)</b> B220 (B-Cells), <b>(e-f)</b> F480 (macrophages), <b>(g-h)</b> CD11c (dendritic cells), and <b>(i- j)</b> Gr-1 (Neutrophils). Insets indicate an EdU⁺ replicating cell that does not express CD3, B220, F480, CD11c, or Gr-1. Scale bar = 100 μm.</p