Xueling Li

Stage-specific signaling through TGFβ family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.

The generation of insulin-producing β-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFβ family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells

Aims/hypothesisUsing a novel directed differentiation protocol, we recently generated up to 25% insulin-producing cells from human embryonic stem cells (hESCs) (insulin+ cells). At this juncture, it was important to functionally and molecularly characterise these hESC-derived insulin+ cells and identify key differences and similarities between them and primary beta cells.MethodsWe used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INSGFP/w) and an untargeted hESC line (HES2). INSGFP/w allowed efficient identification and purification of GFP-producing (INS:GFP+) cells. Insulin+ cells were examined for key features of adult beta cells using microarray, quantitative PCR, secretion assays, imaging and electrophysiology.ResultsImmunofluorescent staining showed complete co-localisation of insulin with GFP; however, cells were often multihormonal, many with granules containing insulin and glucagon. Electrophysiological recordings revealed variable KATP and voltage-gated Ca2+ channel activity, and reduced glucose-induced cytosolic Ca2+ uptake. This translated into defective glucose-stimulated insulin secretion but, intriguingly, appropriate glucagon responses. Gene profiling revealed differences in global gene expression between INS:GFP+ cells and adult human islets; however, INS:GFP+ cells had remarkably similar expression of endocrine-lineage transcription factors and genes involved in glucose sensing and exocytosis.Conclusions/interpretationINS:GFP+ cells can be purified from differentiated hESCs, providing a superior source of insulin-producing cells. Genomic analyses revealed that INS:GFP+ cells collectively resemble immature endocrine cells. However, insulin+ cells were heterogeneous, a fact that translated into important functional differences within this population. The information gained from this study may now be used to generate new iterations of functioning beta cells that can be purified for transplant.

INS GFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells

Aims/hypothesisWe aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS+) cells derived in vitro.MethodsHomologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS+ cells.ResultsDifferentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INSGFP/w hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS+ cells.Conclusions / interpretationINSGFP/w hESCs are a valuable tool for investigating the nature of early INS+ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS+ cells from differentiating hESCs.

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take ∼1 month.

Endocrine cells develop within pancreatic bud-like structures derived from mouse ES cells differentiated in response to BMP4 and retinoic acid

We have examined factors affecting the in vitro differentiation of Pdx1(GFP/w) ESCs to pancreatic endocrine cells. Inclusion of Bone Morphogenetic Protein 4 (BMP4) during the first four days of differentiation followed by a 24-hour pulse of retinoic acid (RA) induced the formation of GFP(+) embryoid bodies (EBs). GFP expression was restricted to E-cadherin(+) tubes and GFP bright (GFP(br)) buds, reminiscent of GFP(+) early foregut endoderm and GFP(br) pancreatic buds observed in Pdx1(GFP/w) embryos. These organoid structures developed without further addition of exogenous factors between days 5 and 12, suggesting that day 5 EBs contained a template for the subsequent phase of development. EBs treated with nicotinamide after day 12 of differentiation expressed markers of endocrine and exocrine differentiation, but only in cells within the GFP(br) buds. Analysis of Pdx1(GFP/w) ESCs modified by targeting a dsRed1 gene to the Ins1 locus (Pdx1(GFP/w)Ins1(RFP/w) ESCs) provided corroborating evidence that insulin positive cells arose from GFP(br) buds, mirroring the temporal relationship between pancreatic bud development and the formation of endocrine cells in the developing embryo. The readily detectable co-expression of GFP and RFP in grafts derived from transplanted EBs demonstrated the utility of Pdx1(GFP/w)Ins1(RFP/w) ESCs for investigating pancreatic differentiation in vitro and in vivo.

The quest for the perfect gravity anomaly: Part 2 - Mass effects and anomaly inversion

Gravity anomalies have become an important tool for geologic studies since the widespread use of high-precision gravimeters after the Second World War. More recently the development of instrumentation for airborne gravity observations, procedures for acquiring data from satellite platforms, the readily available Global Positioning System for precise vertical and horizontal control, improved global data bases, and enhancement of computational hardware and software have accelerated the use of the gravity method. As a result, efforts are being made to improve the gravity databases that are made available to the geoscience community by broadening their observational holdings and increasing the accuracy and precision of the included data. Currently the North American Gravity Database as well as the individual databases of Canada, Mexico, and the United States of America are being revised using new formats and standards. The objective of this paper is to describe the use of the revised standards for gravity data processing and modeling and there impact on geological interpretations.

The quest for the perfect gravity anomaly: Part 1 — New calculation standards

Summary The North American gravity database together with databases from Canada, Mexico, and the United States are being revised to improve their coverage, versatility, and accuracy. An important part of this effort is revision of procedures and standards for calculating gravity anomalies taking into account our enhanced computational power, modern satellite-based positioning technology, improved terrain databases, and increased interest in more accurately defining different anomaly components. The most striking revision is the use of one single internationally accepted reference ellipsoid for the horizontal and vertical datums of gravity stations as well as for the computation of the theoretical gravity. The new standards hardly impact the interpretation of local anomalies, but do improve regional anomalies. Most importantly, such new standards can be consistently applied to gravity database compilations of nations, continents, and even the entire world.

Temporal Restriction of Pancreatic Branching Competence During Embryogenesis Is Mirrored In Differentiating Embryonic Stem Cells

To develop methods for the generation of insulin-producing β-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1(GFP/w) ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP(+) cells formed GFP bright (GFP(br)) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFP(br) buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1(GFP/w) embryos and ESC-derived GFP(br) buds reveal a similar process of proliferation and branching, with both embryonic Pdx1(GFP/w) branching pancreatic epithelium and ESC-derived GFP(br) branching organoids expressing markers representing epithelial (EpCAM and E-Cadherin), ductal (Mucin1), exocrine (Amylase and Carboxypeptidase 1A), and endocrine cell types (Glucagon and Somatostatin). ESC-derived branching structures also expressed a suite of genes indicative of ongoing pancreatic differentiation, paralleling gene expression within similar structures derived from the E13.5 fetal pancreas. In summary, differentiating mouse ESCs can generate pancreatic material that has significant similarity to the fetal pancreatic anlagen, providing an in vitro platform for investigating the cellular and molecular mechanisms underpinning pancreatic development.

Pancreas differentiation of mouse ES cells.

This unit describes the derivation of pancreatic cells from mouse embryonic stem cells (ESCs). Mouse ESCs are pluripotent immortal cells derived from the inner cell mass of pre-implantation blastocyst-stage embryos that possess the ability to differentiate into any cell type within the adult animal. In vitro, ESCs can be differentiated into a variety of cell types representing derivatives of the three embryonic germ layers, mesoderm, endoderm, and ectoderm. Successfully differentiating ES cells to pancreatic cells has the potential to provide an alternative to cadaver-derived cells for treatment of type I diabetes. This unit outlines a method for the differentiation of ESCs toward pancreatic endoderm in serum-free medium from embryoid bodies (EBs) formed in suspension or spin EBs. In addition there is a protocol for maintaining ESC.