Activin A promotes hematopoietic fated mesoderm development through upregulation of brachyury in human embryonic stem cells.

The development of the hematopoietic system involves multiple cellular steps beginning with the formation of the mesoderm from the primitive streak, followed by emergence of precursor populations that become committed to either the endothelial or hematopoietic lineages. A number of growth factors such as activins and fibroblast growth factors (FGFs) are known to regulate the early specification of hematopoietic fated mesoderm, notably in amphibians. However, the potential roles of these factors in the development of mesoderm and subsequent hematopoiesis in the human have yet to be delineated. Defining the cellular and molecular mechanisms by which combinations of mesoderm-inducing factors regulate this stepwise process in human cells in vitro is central to effectively directing human embryonic stem cell (hESC) hematopoietic differentiation. Herein, using hESC-derived embryoid bodies (EBs), we show that Activin A, but not basic FGF/FGF2 (bFGF), promotes hematopoietic fated mesodermal specification from pluripotent human cells. The effect of Activin A treatment relies on the presence of bone morphogenetic protein 4 (BMP4) and both of the hematopoietic cytokines stem cell factor and fms-like tyrosine kinase receptor-3 ligand, and is the consequence of 2 separate mechanisms occurring at 2 different stages of human EB development from mesoderm to blood. While Activin A promotes the induction of mesoderm, as indicated by the upregulation of Brachyury expression, which represents the mesodermal precursor required for hematopoietic development, it also contributes to the expansion of cells already committed to a hematopoietic fate. As hematopoietic development requires the transition through a Brachyury+ intermediate, we demonstrate that hematopoiesis in hESCs is impaired by the downregulation of Brachyury, but is unaffected by its overexpression. These results demonstrate, for the first time, the functional significance of Brachyury in the developmental program of hematopoietic differentiation from hESCs and provide an in-depth understanding of the molecular cues that orchestrate stepwise development of hematopoiesis in a human system.

Monitoring the survival of islet transplants by MRI using a novel technique for their automated detection and quantification.

OBJECT: There is a clinical need to be able to assess graft loss of transplanted pancreatic islets (PI) non-invasively with clear-cut quantification of islet survival. We tracked transplanted PI in diabetic mice during the early post-transplant period by magnetic resonance imaging (MRI) and quantified the islet loss using automatic segmentation technique. MATERIALS AND METHODS: Magnetically labeled islet iso-, allo- and xenografts were injected into the right liver lobes. Animals underwent MRI scanning during 14 days after PI transplantation. MR images were processed using custom-made software, which automatically detects hypointense regions representing PI. It is based on morphological top-hat and bottom-hat transforms. RESULTS: Manually and automatically detected areas, corresponding to PI, differed by 4% in phantoms. Signal loss regions due to PI decreased comparably in all groups during the first week post transplant. Throughout the second week post-transplant, the signal loss area continued in a steep decline in case of allografts and xenografts, whereas the decline in case of isografts slowed down. CONCLUSION: Automatic segmentation allows for the more reproducible, objective assessment of transplanted PI. Quantification confirms the assumption that a significant number of islets are destroyed in the first week following transplantation irrespective of allografts, xenografts or isografts.

IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro.

Distinctive properties of stem cells are not autonomously achieved, and recent evidence points to a level of external control from the microenvironment. Here, we demonstrate that self-renewal and pluripotent properties of human embryonic stem (ES) cells depend on a dynamic interplay between human ES cells and autologously derived human ES cell fibroblast-like cells (hdFs). Human ES cells and hdFs are uniquely defined by insulin-like growth factor (IGF)- and fibroblast growth factor (FGF)-dependence. IGF 1 receptor (IGF1R) expression was exclusive to the human ES cells, whereas FGF receptor 1 (FGFR1) expression was restricted to surrounding hdFs. Blocking the IGF-II/IGF1R pathway reduced survival and clonogenicity of human ES cells, whereas inhibition of the FGF pathway indirectly caused differentiation. IGF-II is expressed by hdFs in response to FGF, and alone was sufficient in maintaining human ES cell cultures. Our study demonstrates a direct role of the IGF-II/IGF1R axis on human ES cell physiology and establishes that hdFs produced by human ES cells themselves define the stem cell niche of pluripotent human stem cells.