Stephen Dang

Controlled, scalable embryonic stem cell differentiation culture.

Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high‐cell‐density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E‐cadherin‐mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell‐cell interactions in scalable culture by the mass encapsulation of ES cells in size‐specified agarose capsules. When placed in stirred‐suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.

In vitro degradation of a novel poly(lactide-co-glycolide) 75/25 foam

Macroporous poly(lactide-co-glycolide) PLGA 75/25 foams were prepared for application in bone tissue engineering. Their in vitro degradation behaviour was followed over a 30 week period at 37 degrees C and at one of three pHs: (1) pH 5.0, which mimics the acidic environment produced by activated macrophages, (2) pH 7.4, which reproduces normal physiological conditions and (3) an intermediate pH 6.4. The degradation of the PLGA 75/25 foams was studied by measuring changes in mass, molecular weight and morphology. The degradation profile of foams maintained at pH 5.0, 6.4 and 7.4 was similar until week 16, after which foams maintained at pH 6.4 and 7.4 had comparable degradation patterns whereas foams maintained at pH 5.0 degraded faster. For example, mass loss was less than 3% for foams maintained at all three pHs until week 16; however, by week 30, foams maintained at pH 6.4 and 7.4 had lost 30% of their mass whereas foams maintained at pH 5.0 had lost 90% of their mass. Foams maintained at pH 6.4 and 7.4 showed a similar constant decrease in molecular weight over the entire degradation study. Foams maintained at pH 5.0 had a similar rate of molecular weight loss as those maintained at pH 6.4 and 7.4 until week 16, after which the rate of molecular weight loss of foams maintained at pH 5.0 was accelerated. The morphology of the foams maintained at pH 6.4 and 7.4 was unchanged for 25 weeks. Foams maintained at pH 5.0 collapsed after week 18. Thus the PLGA 75/25 foams, described herein, maintained their 3-D morphology at physiological pH for over 6 months, which is an important feature for tissue engineering applications.

Soluble Flt-1 regulates Flk-1 activation to control hematopoietic and endothelial development in an oxygen-responsive manner.

Vascular endothelial growth factor (VEGF) and the vascular endothelial growth factor receptors (VEGFRs) regulate the development of hemogenic mesoderm. Oxygen concentration‐mediated activation of hypoxia‐inducible factor targets such as VEGF may serve as the molecular link between the microenvironment and mesoderm‐derived blood and endothelial cell specification. We used controlled‐oxygen microenvironments to manipulate the generation of hemogenic mesoderm and its derivatives from embryonic stem cells. Our studies revealed a novel role for soluble VEGFR1 (sFlt‐1) in modulating hemogenic mesoderm fate between hematopoietic and endothelial cells. Parallel measurements of VEGF and VEGFRs demonstrated that sFlt‐1 regulates VEGFR2 (Flk‐1) activation in both a developmental‐stage‐dependent and oxygen‐dependent manner. Early transient Flk‐1 signaling occurred in hypoxia because of low levels of sFlt‐1 and high levels of VEGF, yielding VEGF‐dependent generation of hemogenic mesoderm. Sustained (or delayed) Flk‐1 activation preferentially yielded hemogenic mesoderm‐derived endothelial cells. In contrast, delayed (sFlt‐1‐mediated) inhibition of Flk‐1 signaling resulted in hemogenic mesoderm‐derived blood progenitor cells. Ex vivo analyses of primary mouse embryo‐derived cells and analysis of transgenic mice secreting a Flt‐1‐Fc fusion protein (Fc, the region of an antibody which is constant and binds to receptors) support a hypothesis whereby microenvironmentally regulated blood and endothelial tissue specification is enabled by the temporally variant control of the levels of Flk‐1 activation.