Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population

In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.     

Markers of cell lineage, differentiation and activation

The most widespread use of CD markers is in the determination of cell lineage and sublineage. For example, T cells are identified by the expression of CD3 (reviewed in this issue of CD corner). A mature T cell may belong to the T4 subset, in which case it will express CD4. Similarly, there are markers for other cell populations and sub-populations. Within the lineages, it is helpful to distinguish cells at different stages of differentiation and activation. Differentiation status is particularly useful in the diagnostic analysis of the lymphoid and myeloid malignancies, and in research on the haemopoietic system. Examples include markers for na?ve or antigen-experienced cells (especially the CD45 isoforms) and molecules such as CALLA (CD9) found on B-lineage precursors, including B lineage acute lymphoblastic leukaemia. Activation status is especially interesting in studies of cell function. Activation markers include growth factor receptors such as CD25 (a component of the receptor for IL-2), and molecules who's cellular function is not fully understood, such as CD69 and CD98. These markers have revolutionised aspects of pathology and research, and the ease with which some cell populations can be identified has lead to some unrealised, and perhaps unrealistic, expectations. We expect to be able to identify T helper type 1 (TH1) and T helper type 2 (TH2) cells on the basis of a simple surface marker; we are frustrated by the lack of a single marker for all dendritic cells or for all NK cells; we are confused by the un-coordinated expression of different activation markers; we tend to over-interpret phenotype in some situations. To find solutions to these problems it is helpful to examine why the successful lineage markers work so well, and to reconsider our expectations. Lineage markers are the main focus in this commentary; the question of activation markers and markers of differentiation state will be considered in a separate paper.     

Correlation between cell morphology and aggrecan gene expression level during differentiation from mesenchymal stem cells to chondrocytes

A morphological parameter of polygonal index was defined as the ratio of cell adhesion area versus the square of the major cell axis, and cells that had an adhesion area larger than 4000 mum(2) and a polygonal index larger than 0.3 were considered large polygonal cells. Cell morphology tended to change from fibroblast-like to polygonal and the percentage of the large polygonal cells increased almost in proportion to aggrecan mRNA expression level during the differentiation culture of mesenchymal stem cells (MSCs) to chondrocytes. Approximately 80% of the large polygonal cells were negative for MSC marker (CD90, CD166) expression and the aggrecan mRNA expression level of the large polygonal cells was markedly higher than that of cells with other morphologies.     

Mesodermal cell lineage determination and proto-oncogene expression

Previously, we have established a rat embryo fibroblastic cell line that is characterized as immortal, nontumorigenic and retains a diploid karyotype. We report here that this cell line will spontaneously differentiate along mesodermal cell lineages to form myotubes and adipocytes. We have isolated and characterized lineage-determined (nondifferentiated) preadipocytes and myoblasts. Using these cell lines we have investigated the expression of proto-oncogenes concomitant with lineage-specific determination. No major changes in proto-oncogene RNA levels were observed that correlated with lineage determination. These results would suggest that none of the 15 proto-oncogenes used in these experiments are involved in mesodermal lineage determination; but this does not rule out the possibility that other proto-oncogenes not tested or currently unknown may be involved. However, these differentiating subclones and nondifferentiating fibroblasts that exhibit a normal karyotype will be an excellent model-system to investigate the molecular basis of cell lineage determination.     

Self-organization vs Watchmaker: stochastic gene expression and cell differentiation

Cell differentiation and organism development are traditionally described in deterministic terms of program and design, echoing a conventional clockwork perception of the cell on another scale. However, the current experimental reality of stochastic gene expression and cell plasticity is poorly consistent with the ideas of design, purpose and determinism, suggesting that the habit of classico-mechanistic interpretation of life phenomena may handicap our ability to adequately comprehend and model biological systems. An alternative conceptualization of cell differentiation and development is proposed where the developing organism is viewed as a dynamic self-organizing system of adaptive interacting agents. This alternative interpretation appears to be more consistent with the probabilistic nature of gene expression and the phenomena of cell plasticity, and is coterminous with the novel emerging image of the cell as a self-organizing molecular system. I suggest that stochasticity, as a principle of differentiation and adaptation, and self-organization, as a concept of emergence, have the potential to provide an interpretational framework that unites phenomena across different scales of biological organization, from molecules to societies.Edited by R.J. Sommer