Differentiation of human embryonic stem cells (hescs) toward cells with neural charectristics

Zusammenfassung

First Part: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. These findings suggest that our differentiation protocol has the capacity to generate region-specific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-culture with stromal cells and without presence of cells of mesodermal or endodermal lineages. Seccond part: The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell (hESC) differentiation into cerebellar neurons. Derived cells showed T-shaped polarity phenotype expressing similar markers as developed human cerebellum. Electrophysiological measurements confirmed functional electric properties compatible with these cells. In vivo implantation of differentiated hESC transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular, the Purkinje cell and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in development of cerebellum can be efficiently recapitulated in vitro which enables design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases.