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Many patients with type 1 diabetes (T1D) have residual β cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual β cells and α cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.
Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Human pluripotent stem cells (hPSCs) maintain a highly fragmented mitochondrial network, but the mechanisms regulating this phenotype remain unknown. Here, we describe a non-cell death function of the anti-apoptotic protein, MCL-1, in regulating mitochondrial dynamics and promoting pluripotency of stem cells. MCL-1 is induced upon reprogramming, and its inhibition or knockdown induces dramatic changes to the mitochondrial network as well as loss of the key pluripotency transcription factors, NANOG and OCT4. Aside from localizing at the outer mitochondrial membrane like other BCL-2 family members, MCL-1 is unique in that it also resides at the mitochondrial matrix in pluripotent stem cells. Mechanistically, we find MCL-1 to interact with DRP-1 and OPA1, two GTPases responsible for remodeling the mitochondrial network. Depletion of MCL-1 compromised the levels and activity of these key regulators of mitochondrial dynamics. Our findings uncover an unexpected, non-apoptotic function for MCL-1 in the maintenance of mitochondrial structure and stemness.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Direct reprogramming of fibroblasts into cardiomyocytes is a promising approach for cardiac regeneration but still faces challenges in efficiently generating mature cardiomyocytes. Systematic optimization of reprogramming protocols requires scalable, objective methods to assess cellular phenotype beyond what is captured by transcriptional signatures alone. To address this question, we automatically segmented reprogrammed cardiomyocytes from immunofluorescence images and analyzed cell morphology. We also introduce a method to quantify sarcomere structure using Haralick texture features, called SarcOmere Texture Analysis (SOTA). We show that induced cardiac-like myocytes (iCLMs) are highly variable in expression of cardiomyocyte markers, producing subtypes that are not typically seen in vivo. Compared to neonatal mouse cardiomyocytes, iCLMs have more variable cell size and shape, have less organized sarcomere structure, and demonstrate reduced sarcomere length. Taken together, these results indicate that traditional methods of assessing cardiomyocyte reprogramming by quantifying induction of cardiomyocyte marker proteins may not be sufficient to predict functionality. The automated image analysis methods described in this study may enable more systematic approaches for improving reprogramming techniques above and beyond existing algorithms that rely heavily on transcriptome profiling.
Beta cell replacement strategies hold promise for permanently treating type 1 diabetes. In Cell Stem Cell, Xiao et al. (2018) restore pancreatic beta cell mass and normalize blood glucose in diabetic mice by reprogramming pancreatic alpha to beta cells using Pdx1- and Mafa-expressing adeno-associated virus infused into the pancreatic duct.
Copyright © 2017 Elsevier Inc. All rights reserved.
Tuberous sclerosis complex (TSC) is a pediatric disorder of dysregulated growth and differentiation caused by loss of function mutations in either the TSC1 or TSC2 genes, which regulate mTOR kinase activity. To study aberrations of early development in TSC, we generated induced pluripotent stem cells using dermal fibroblasts obtained from patients with TSC. During validation, we found that stem cells generated from TSC patients had a very high rate of integration of the reprogramming plasmid containing a shRNA against TP53. We also found that loss of one allele of TSC2 in human fibroblasts is sufficient to increase p53 levels and impair stem cell reprogramming. Increased p53 was also observed in TSC2 heterozygous and homozygous mutant human stem cells, suggesting that the interactions between TSC2 and p53 are consistent across cell types and gene dosage. These results support important contributions of TSC2 heterozygous and homozygous mutant cells to the pathogenesis of TSC and the important role of p53 during reprogramming.
© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
Otto Warburg's discovery in the 1920s that tumor cells took up more glucose and produced more lactate than normal cells provided the first clues that cancer cells reprogrammed their metabolism. For many years, however, it was unclear as to whether these metabolic alterations were a consequence of tumor growth or an adaptation that provided a survival advantage to these cells. In more recent years, interest in the metabolic differences in cancer cells has surged, as tumor proliferation and survival have been shown to be dependent upon these metabolic changes. In this educational review, we discuss some of the mechanisms that tumor cells use for reprogramming their metabolism to provide the energy and nutrients that they need for quick or sustained proliferation and discuss the potential for therapeutic targeting of these pathways to improve patient outcomes.
A cell's phenotype is the observable actualization of complex interactions between its genome, epigenome, and local environment. While traditional views in cancer have held that cellular and tumor phenotypes are largely functions of genomic instability, increasing attention has recently been given to epigenetic and microenvironmental influences. Such non-genetic factors allow cancer cells to experience intrinsic diversity and plasticity, and at the tumor level can result in phenotypic heterogeneity and treatment evasion. In 2006, Takahashi and Yamanaka exploited the epigenome's plasticity by "reprogramming" differentiated cells into a pluripotent state by inducing expression of a cocktail of four transcription factors. Recent advances in cancer biology have shown not only that cellular reprogramming is possible for malignant cells, but it may provide a foundation for future therapies. Nevertheless, cell reprogramming experiments are frequently plagued by low efficiency, activation of aberrant transcriptional programs, instability, and often rely on expertise gathered from systems which may not translate directly to cancer. Here, we review a theoretical framework tracing back to Waddington's epigenetic landscape which may be used to derive quantitative and qualitative understanding of cellular reprogramming. Implications for tumor heterogeneity, evolution and adaptation are discussed in the context of designing new treatments to re-sensitize recalcitrant tumors. This article is part of a Special Issue entitled: Evolutionary principles - heterogeneity in cancer?, edited by Dr. Robert A. Gatenby.
Copyright © 2017. Published by Elsevier B.V.
Parietal cell atrophy is considered to cause metaplasia in the stomach. We developed mice that express the diphtheria toxin receptor specifically in parietal cells to induce their death, and found this to increase proliferation in the normal stem cell zone and neck but not to cause metaplastic reprogramming of chief cells. Furthermore, the metaplasia-inducing agents tamoxifen or DMP-777 still induced metaplasia even after previous destruction of parietal cells by diphtheria toxin. Atrophy of parietal cells alone therefore is not sufficient to induce metaplasia: completion of metaplastic reprogramming of chief cells requires mechanisms beyond parietal cell injury or death.
Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.
Artificial transcription factors (ATFs) are precision-tailored molecules designed to bind DNA and regulate transcription in a preprogrammed manner. Libraries of ATFs enable the high-throughput screening of gene networks that trigger cell fate decisions or phenotypic changes. We developed a genome-scale library of ATFs that display an engineered interaction domain (ID) to enable cooperative assembly and synergistic gene expression at targeted sites. We used this ATF library to screen for key regulators of the pluripotency network and discovered three combinations of ATFs capable of inducing pluripotency without exogenous expression of Oct4 (POU domain, class 5, TF 1). Cognate site identification, global transcriptional profiling, and identification of ATF binding sites reveal that the ATFs do not directly target Oct4; instead, they target distinct nodes that converge to stimulate the endogenous pluripotency network. This forward genetic approach enables cell type conversions without a priori knowledge of potential key regulators and reveals unanticipated gene network dynamics that drive cell fate choices.