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

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous 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. RESULTS: Differentiation of INS(GFP/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. INS(GFP/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/INTERPRETATION: INS(GFP/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.

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

AIMS/HYPOTHESIS: Using 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. METHODS: We used a new reporter hESC line with green fluorescent protein (GFP) cDNA targeted to the INS locus by homologous recombination (INS (GFP/w)) and an untargeted hESC line (HES2). INS (GFP/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. RESULTS: Immunofluorescent 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 K(ATP) and voltage-gated Ca(2+) channel activity, and reduced glucose-induced cytosolic Ca(2+) 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/INTERPRETATION: INS: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.

A systems biology approach to Down syndrome: identification of Notch/Wnt dysregulation in a model of stem cells aging.

Stem cells are central to the development and maintenance of many tissues. This is due to their capacity for extensive proliferation and differentiation into effector cells. More recently it has been shown that the proliferative and differentiative ability of stem cells decreases with age, suggesting that this may play a role in tissue aging. Down syndrome (DS), is associated with many of the signs of premature tissue aging including T-cell deficiency, increased incidence of early Alzheimer-type, Myelodysplastic-type disease and leukaemia. Previously we have shown that both hematopoietic (HSC) and neural stem cells (NSC) in patients affected by DS showed signs of accelerated aging. In this study we tested the hypothesis that changes in gene expression in HSC and NSC of patients affected by DS reflect changes occurring in stem cells with age. The profiles of genes expressed in HSC and NSC from DS patients highlight pathways associated with cellular aging including a downregulation of DNA repair genes and increases in proapoptotic genes, s-phase cell cycle genes, inflammation and angiogenesis genes. Interestingly, Notch signaling was identified as a potential hub, which when deregulated may drive stem cell aging. These data suggests that DS is a valuable model to study early events in stem cell aging.

Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications.

The discovery of human pluripotent stem cells has laid the foundation for an emerging new field of biomedical research that holds promise to develop models of human development and disease, establish new strategies for discovering and testing drugs, and provide systems for the generation of cells and tissues for transplantation for the treatment of disease. The remarkable potential of pluripotent stem cells has sparked interest and excitement in academia, the biotechnology and pharmaceutical industries, as well as the lay public. Although the potential of human pluripotent stem cells is truly outstanding, fulfilling this potential is solely dependent on our ability to efficiently generate functional cell types from them. Some of the most successful approaches in this area to date are those that have applied the principles of developmental biology to stem cell differentiation. In this chapter, we review these concepts and highlight specific examples demonstrating that pluripotent stem cell differentiation in culture recapitulates the key aspects of early embryonic development. By continuing to translate insights from embryology to stem cell biology, progress in our ability to generate specific cell types from pluripotent stem cells will advance, yielding enriched populations of human cell types, including cardiomyocytes, hematopoietic cells, hepatocytes, pancreatic beta cells, and neural cells, for drug discovery, functional evaluation in preclinical models of human disease, and ultimately clinical applications.

'Neurographic' palmaris brevis sign in type II degrees ulnar neuropathy at wrist.

OBJECTIVE: To investigate the source of an unusual and previously unreported volume conducted potential on motor nerve conduction studies. In a case of subacute ulnar neuropathy at wrist (UNW) selectively involving the deep motor branch, we recorded from the hypothenar eminence a large positive wave (2.5 ms-2 mV) preceding the negative takeoff of the delayed distal ulnar motor response. METHODS: We performed multiple channels motor and sensory ulnar nerve (UN) conduction studies; these included selective electrical stimulation and anaesthetic block of UN branches and also selective recording of motor responses by single fibre needles; data were confirmed by an intraoperative neurophysiological study and correlated with MRI and surgical findings. RESULTS: Detailed neurophysiological investigation demonstrated the generation of this waveform from the palmaris brevis (PB) muscle. MRI and surgical exploration documented a hypertrophy of this muscle. CONCLUSIONS: In type II degrees UNW, depolarization of a spared palmaris brevis muscle may be recorded as a positive wave preceding the delayed abductor digiti minimi motor response. SIGNIFICANCE: We underline the peculiar localizing value of this volume conducted 'meaningful artefact' in that particular setting. It actually represented an early neurographic analogue of what is known as the clinical 'Palmaris Brevis Sign' in long standing type II degrees UNW.

Bcl-w expression in colorectal adenocarcinoma.

We have found that the anti-apoptotic Bcl-2 family protein, Bcl-w, was frequently expressed in colorectal adenocarcinomas, with 69/75 showing positive staining with anti-Bcl-w IgG. Adenomas demonstrated a much lower frequency of Bcl-w expression (only 1 of 17), as did adenocarcinomas from other epithelial tissues such as breast (0/8), stomach (1112) and cervix (0/12). Bcl-w status could be related to the histopathological classification of the tumours, with TNM stage III tumours showing significantly higher levels of expression than tumours of better prognostic grade (at P = 0.009). Those patients with node involvement also had tumours with significantly elevated levels of Bcl-w (at P = 0.02), compared to those which were node-negative. The results suggest that Bcl-w could play a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Currently, more data are being collected to allow us to assess the importance of Bcl-w for disease progression and patient survival.