[show abstract][hide abstract] ABSTRACT: Here, we describe the generation of viable and dopamine-producing neurons derived from pluripotent mouse embryonic stem cells. Neurotrophic factors in combination with survival-promoting factors, such as interleukin-1beta, glial cell line-derived neurotrophic factor, neurturin, transforming growth factor-beta(3) and dibutyryl-cyclic AMP, significantly enhanced Nurr1 and tyrosine hydroxylase (TH) mRNA levels, whereas En-1, mash-1 and dopamine-2-receptor mRNA levels were not upregulated. In parallel, mRNA levels of the anti-apoptotic gene bcl-2 were found to be upregulated at terminal stages. Double immunofluorescence analysis revealed increased numbers of TH- and dopamine transporter-, but not gamma-aminobutyric acid- and serotonin-positive neurons in relation to synaptophysin-labeled cells by survival-promoting factors. Moreover, high-performance liquid chromatography analysis showed detectable levels of intracellular dopamine. We conclude that survival-promoting factors enhance differentiation, survival and maintenance of dopaminergic neurons derived from embryonic stem cells.
Mechanisms of Development 08/2001; 105(1-2):93-104. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: beta(1)integrin-deficient (beta(1)-/-) ES cells showed increased differentiation of cardiac cells characterized by reduced adhesion and high beating frequency. Whereas in whole embryoid body outgrowths of beta(1)-/- cells maximum levels of alpha(v), beta(3)and beta(5)integrin mRNA were delayed and transiently upregulated, in cardiac clusters isolated from beta(1)-/- cells, only beta(3)integrin mRNA levels were enhanced in comparison to wild-type (wt) cells. To answer the question, whether alpha(v)and beta(3)integrins may compensate, at least partially, the loss of beta(1)integrin function during cardiac differentiation, the distribution of alpha(v)and beta(3)integrins in beta(1)-/- and wt pacemaker-like cardiac cells was analyzed. A different distribution of alpha(v)and beta(3)integrins in beta(1)-/- v wt cardiac cells was found. In wt cardiac cells, beta(1)integrin was localized in specialized subsarcolemmal regions, in particular, at focal contacts and costameres, but alpha(v)integrin was diffusely distributed. In contrast, in beta(1)-/- cardiac cells, alpha(v)integrin was preponderantly localized at cell membranes, focal contacts and costameres. beta(3)integrin displayed a diffuse pattern both in wt and in beta(1)-/- pacemaker-like cells at early differentiation stages, whereas at terminal stages, beta(3)was colocalized with sarcomeres in wt, but not in beta(1)-/- pacemaker-like cells. Quantitative immunofluorescence analysis revealed increased alpha(v)and beta(3)integrin levels in beta(1)-/- pacemaker-like cardiac cells. Our results led us to conclude that altered cellular distribution of alpha(v)integrin and upregulation of beta(3)integrin correlate with growth and survival of beta(1)-/- cardiac pacemaker-like cells at an early developmental state. However, alpha(v)and beta(3)integrins cannot functionally compensate the loss of beta(1)integrin during terminal differentiation of cardiac cells implicating that cardiomyocytes require specific beta(1)integrin functions for cardiac specialization.
Journal of Molecular and Cellular Cardiology 04/2001; 33(3):521-32. · 5.15 Impact Factor