Archive for the ‘stem cells’ Category
Mouse ES Cell Experiments
Posted by admin in stem cells on June 21st, 2009
Signal-Driven Approaches
Based on the conservation between mouse and human pancreatic development, the first attempts at differentiating ES cells into pancreatic endocrine beta cells were done using mouse cells, given their relative ease of use. One of the first efforts was led by Lumelsky and coworkers, who adapted a neural differentiation protocol to murine ES cells and reported the generation of insulin-secreting structures. The rationale behind the approach has its roots back in the 1980s, when the prevailing view was that islet precursor cells were neurons. This assumption was made based on observations such as the Read the rest of this entry »
Embryonic Stem Cells and Pancreatic Differentiation
Posted by admin in stem cells on June 21st, 2009
Embryonic stem (ES) cells are derived from the early preimplantation blastocyst. These cells are immortal under defined conditions in vitro, and can be indefinitely expanded without loss of pluripotency. Proof-of-concept experiments demonstrate that they have the ability to spontaneously differentiate into insulin-producing cells, even if at a very low frequency. Here we review the most recent progress at defining conditions (chemical, genetic, or otherwise) for the directed differentiation of both mouse and human ES cells into insulinproducing beta cells. Read the rest of this entry »
In Vivo
Posted by admin in stem cells on June 21st, 2009
The basic principles of tissue culture have evolved little since the 1940s. With some exceptions, most culture devices are variations on the common theme of a plastic container that holds the cells and their culture medium. Despite the progress in our understanding of the microenvironmental regulation of pancreatic development, it is unlikely that we will ever be able to reproduce in vitro the exquisite complexity of the native niche where beta cells develop. These limitations are quite possibly the main reason why most in vitro differentiation protocols are inefficient, yielding only small percentages of the desired cell types. Read the rest of this entry »
Microenvironment
Posted by admin in stem cells on June 21st, 2009
It could be reasoned that the in vitro specification of stem cells along the beta cell lineage would require the accurate recapitulation of the differentiation steps by providing the cells with the chemical cues that are known to sequentially activate critical transcription factors. Read the rest of this entry »
Protein Transduction
Posted by admin in stem cells on June 21st, 2009
The general scheme of gene-based differentiation toward pancreatic endocrine cells would be to transfect stem cells, in a stepwise fashion, with candidate pro-endodermal, pro-pancreatic, and pro-endocrine expression vectors. However, a conventional strategy for transfection/antibiotic selection of clones would be extremely difficult to reduce to practice, as every step would require the selection of clones containing the appropriate integration. Since ES cells tend to progressively stop dividing after the initiation of differentiation, repeating this process for each one of the subsequent genes would be nearly impossible. As for the use of viruses to deliver these genes, we have already stated the problems that will likely stand in the way of the development of clinical therapies. Read the rest of this entry »
Genetic Manipulation
Posted by admin in stem cells on June 21st, 2009
The problems of chemical differentiation could be circumvented, at least in theory, by sequentially transfecting huES cells with the genes encoding for the key transcription factors whose activation is known to choreograph pancreatic development. With this idea in mind, many groups around the world have used a number of vectors to deliver active cassettes to stem cells of all origins, including Pdx1, Pax4, Foxa2, Ngn3, NeuroD and many others. Among the vectors, adenoviruses have been highly favored due to the fact that they infect both dividing and nondiving cells, and usually do not integrate in a permanent manner into the genome. Retroviruses, in contrast, have a preference for Read the rest of this entry »
Stem Cell Differentiation: General Approaches
Posted by admin in stem cells on June 21st, 2009
Regardless of the cell type used as a building block for differentiation/ transdifferentiation into pancreatic cells, there are only a few strategies that can be used to modify, both in vitro and in vivo, their fate and behavior. Conventional approaches are based on the addition of chemical soluble agents to the culture medium (signal-driven strategies), in an attempt to mimic the complex symphony of differentiation/specification factors that drive the process in vivo. Extracellular matrices and cell growth substrates may help increase the overall efficiency of these methods. Alternatively, external signaling can be bypassed by means of adding constitutively activated copies of key transcription factors or – more recently – cell-permeable proteins. The rationale of in vivo differentiation is that only the recipient’s body can provide developing cells with the adequate microenvironment to support terminal maturation. Read the rest of this entry »
Reignition of the Embryonic Developmental Program
Posted by admin in stem cells on June 21st, 2009
The field of beta cell regeneration is evolving so rapidly that new discoveries are challenged even before they become mainstream. For many years, it was conventional wisdom that adult beta cell regeneration took place from the ductal system, with only morphological evidence of a recapitulation of embryonic development. The observation that – at least in the mouse – adult beta cell turnover/ regeneration was due to self-replication rather than stem cell differentiation was a complete paradigm shift. Just when this novel notion was starting to settle in the collective understanding of the field, it was challenged again by both additional lineage tracing experiments showing ductal contribution to islet regeneration and the unexpected finding that, under Read the rest of this entry »
Molecular Mechanisms of Islet Regeneration
Posted by admin in stem cells on June 21st, 2009
Reversible Epithelial-to-Mesenchymal Transition
The epithelial–mesenchymal transition (EMT) is a well-known developmental phenomenon by which epithelial cell types (which are typically arrayed in polarized cellular sheets with tight junctions that restrict the movement of their individual components) undergo a series of molecular changes that result in their progressive transformation into mesenchymal cells (characterized by a reduction of cell-to-cell adherence and enhanced migratory capacity). This process is defined by the down-regulation of epithelial proteins (E-cadherin, cytokeratins, occluding, desmoplakin) and the upregulation of mesenchymal ones (N-cadherin, vimentin, fibronectin). Transcription factors such as Snail 1, Snail2/Slug, Twist, and others will inhibit E-cadherin expression and promote EMT. Originally described by Hay more than a decade ago, this transition has been observed both during embryogenesis and in malignant transformation . Read the rest of this entry »
Do New Beta Cells Arise from the Duct/Acinar Tissue
Posted by admin in stem cells on June 21st, 2009
Virtually all researchers on pancreatic and islet cell biology are familiar with the occasional sight of a single islet cell or small islets seemingly sprouting from the ducts of a section of adult pancreas. The incidence of such observations is amplified under a number of experimental or pathological conditions. For a long time, the obvious conclusion has been that islets might indeed be formed in or near the ducts, migrating at a later point to the acinar surroundings. BrdU labeling studies led to Bonner-Weir and colleagues to hypothesize that pancreatic regeneration in the partially pancreatectomized rat occurs through two pathways, namely: the self-replication of existing endocrine and exocrine cells; and the proliferation and differentiation of the ductal epithelium into new Read the rest of this entry »