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Articles by Hsun Teresa Ku in JoVE
JoVE General
Assay כמותי עבור אינסולין להביע המושבה יוצרי אבות
1Department of Biotechnology & Bioinformatics, California State University Channel Islands, 2Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, 3The Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope
Assay תלת מימדי המאפשר clonogenic הלבלב כמו אבות להתמיין אינסולין להביע המושבות מתואר. שיטה זו מנצלת חצי מוצק מדיה המכילה גורמים, methylcellulose Matrigel וצמיחה, שבו אבות יחיד להתרבות ולהבחין
Other articles by Hsun Teresa Ku on PubMed
Committing Embryonic Stem Cells to Early Endocrine Pancreas in Vitro
A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm-derived pancreas and to distinguish insulin-expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES-derived embryoid bodies cultured in the presence of stage-specific concentrations of monothio-glycerol and 15% fetal calf serum, followed by serum-free conditions, give rise to a population that expresses insulin I, insulin II, pdx-1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C-peptide. Most, but not all, of the insulin+ or C-peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of beta-cell specification and differentiation factors activin beta B, nicotinamide, and exendin-4 to later-stage culture increased insulin-positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin-staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.
Insulin-expressing Colonies Developed from Murine Embryonic Stem Cell-derived Progenitors
Previous studies describe a unique culture method for the commitment of murine embryonic stem cells to early endocrine pancreata. In this report, early pancreatic-like beta-cell progenitors were enriched and a colony assay devised to allow these progenitors to differentiate into insulin-expressing colonies in vitro. An embryonic stem cell line with enhanced green fluorescent protein (EGFP) inserted into one allele of neurogenin 3 (Ngn3), a marker for pancreatic endocrine progenitors, was differentiated. During the late stage of culture, 20-30% of cells were Ngn3-EGFP(+). Gene expression profiling using the PancChip microarray platform demonstrated that Ngn3-EGFP(+) cells differentially express endocrine-related genes. A novel semisolid culture method was developed to support the formation of individual insulin/C-peptide-expressing colonies from dissociated single cells. Approximately 0.1-0.6% of Ngn3-EGFP(+) cells gave rise to insulin-expressing colonies, a three- to fivefold enrichment of beta-cell-like progenitors, or insulin-expressing colony-forming units (ICFUs), compared with nonsorted cells. All of the single colonies expressed insulin II, while 69% coexpressed insulin I and 44% coexpressed glucagon. Some single colonies expressed insulin I, insulin II, and Pdx-1 (pancreatic duodenal homeobox-1), but not glucagon. In other colonies, glucagon expression overlapped with C-peptide II in double immunostaining analysis, suggesting heterogeneity among the ICFUs and their resulting colonies. Together, these results demonstrate that progenitors that have the potential to give rise to insulin-expressing cells can be derived from murine embryonic stem cells.
Minireview: Pancreatic Progenitor Cells--recent Studies
Past studies of pancreatic progenitor cell biology relied mostly on histological analyses. Recent studies, using genetic labeling and tracing of progenitors, direct single cell analyses, colony assays, and enrichment of the minor population of progenitor cells through the use of cell surface markers, have strongly suggested that pancreatic progenitor cells with various frequency and lineage potentials, including the multipotent progenitors that give rise to endocrine, exocrine, and duct cells, exist in the developing and adult pancreas. In this review, it is therefore proposed that pancreatic progenitor cells may be organized in a hierarchy, in which the most primitive pan-pancreatic multipotent progenitors are at the top and rare, and the monopotent progenitors are at the bottom and abundant. This model may explain why only drastic injuries lead to effective activation of the progenitor cell compartment of the higher hierarchy, whereas under steady state, pregnancy, and milder injuries, recruitment of preexisting mature cells or their immediate monopotent progenitors could be sufficient to restore metabolic homeostasis. It is also proposed that the morphologically defined ductal cells are likely to be functionally heterogeneous and that endocrine progenitor cell activity should be determined based on functional analyses rather than histological locations.
Insulin Gene Expression is Regulated by DNA Methylation
Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression.
Characterization of an in Vitro Differentiation Assay for Pancreatic-like Cell Development from Murine Embryonic Stem Cells: Detailed Gene Expression Analysis
Embryonic stem (ES) cell technology may serve as a platform for the discovery of drugs to treat diseases such as diabetes. However, because of difficulties in establishing reliable ES cell differentiation methods and in creating cost-effective plating conditions for the high-throughput format, screening for molecules that regulate pancreatic beta cells and their immediate progenitors has been limited. A relatively simple and inexpensive differentiation protocol that allows efficient generation of insulin-expressing cells from murine ES cells was previously established in our laboratories. In this report, this system is characterized in greater detail to map developmental cell stages for future screening experiments. Our results show that sequential activation of multiple gene markers for undifferentiated ES cells, epiblast, definitive endoderm, foregut, and pancreatic lineages was found to follow the sequence of events that mimics pancreatic ontogeny. Cells that expressed enhanced green fluorescent protein, driven by pancreatic and duodenal homeobox 1 or insulin 1 promoter, correctly expressed known beta cell lineage markers. Overexpression of Sox17, an endoderm fate-determining transcription factor, at a very early stage of differentiation (days 2-3) enhanced pancreatic gene expression. Overexpression of neurogenin3, an endocrine progenitor cell marker, induced glucagon expression at stages when pancreatic and duodenal homeobox 1 message was present (days 10-16). Forced expression (between days 16 and 25) of MafA, a pancreatic maturation factor, resulted in enhanced expression of insulin genes, glucose transporter 2 and glucokinase, and glucose-responsive insulin secretion. Day 20 cells implanted in vivo resulted in pancreatic-like cells. Together, our differentiation assay recapitulates the proceedings and behaviors of pancreatic development and will be valuable for future screening of beta cell effectors.
