Atsushi Kubo

Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation

The hematopoietic and endothelial lineages derive from mesoderm and are thought to develop through the maturation of a common progenitor, the hemangioblast. To investigate the developmental processes that regulate mesoderm induction and specification to the hemangioblast, we generated an embryonic stem cell line with the green fluorescent protein (GFP) targeted to the mesodermal gene, brachyury. After the in vitro differentiation of these embryonic stem cells to embryoid bodies, developing mesodermal progenitors could be separated from those with neuroectoderm potential based on GFP expression. Co-expression of GFP with the receptor tyrosine kinase Flk1 revealed the emergence of three distinct cell populations, GFP-Flk1-, GFP+Flk1- and GFP+Flk1+ cells, which represent a developmental progression ranging from pre-mesoderm to prehemangioblast mesoderm to the hemangioblast.

Committing Embryonic Stem Cells to Early Endocrine Pancreas In Vitro

A panel of genetic markers was used to assess the in vitro commitment of murine embryonic stem (ES) cells toward the endoderm‐derived pancreas and to distinguish insulin‐expressing cells of this lineage from other lineages such as neuron, liver, and yolk sac. There are two nonallelic insulin genes in mice. Neuronal cells express only insulin II, whereas the pancreas expresses both insulin I and II. Yolk sac and fetal liver express predominately insulin II, small amounts of insulin I, and no glucagon. We found that ES‐derived embryoid bodies cultured in the presence of stage‐specific concentrations of monothio‐glycerol and 15% fetal calf serum, followed by serum‐free conditions, give rise to a population that expresses insulin I, insulin II, pdx‐1 (a pancreas marker), and Sox17 (an endoderm marker). Immunohistochemical staining shows intracellular insulin particles, and its de novo production was confirmed by staining for C‐peptide. Most, but not all, of the insulin+ or C‐peptide+ cells coexpress glucagon, demonstrating a differentiation pathway to pancreas rather than yolk sac or fetal liver. Addition of β‐cell specification and differentiation factors activin β B, nicotinamide, and exendin‐4 to later‐stage culture increased insulin‐positive cells to 2.73% of the total population, compared with the control culture, which gave rise to less than 1% insulin‐staining cells. These findings suggest that stepwise culture manipulations can direct ES cells to become early endocrine pancreas.

The heart LIM protein gene (Hlp), expressed in the developing and adult heart, defines a new tissue-specific LIM-only protein family

In a subtraction designed to identify transcripts accompanying mesodermal lineage specification in mouse ES differentiation cultures, we identified a gene encoding a two LIM-domain protein which we named heart LIM protein (Hlp). Hlp is most closely related to thymus LIM protein, and these two genes comprise a new gene family related to the cysteine-rich protein (CRP) gene family. In the embryo, Hlp expression is primarily restricted to the developing heart. In situ hybridization showed expression at E7.75 in the paired heart-forming primordia prior to linear heart-tube formation. At E8.5, strong expression is detected in the heart, with equal expression in both heart chambers. Hlp expression is detected in both myocardium and endocardium, and in vascular endothelium. Later in fetal development low levels of Hlp expression are detected outside the heart, including dorsal root ganglia and the spinal cord. In the adult, Hlp is expressed at highest levels in the heart, and at lower levels in the brain, skeletal muscle and aorta. Hlp expression is unchanged in hypertrophic hearts induced by aortic constriction. These data suggest a role for the two LIM-domain gene Hlp in the very earliest stages of heart differentiation and development.