Scientific Resources

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study,we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors,by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner,which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors,in a 2-dimensional or 3-dimensional environment. However,Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices,we observed mature Purkinje-like cells with right morphology and marker expression patterns,which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases. View Publication

Efficient derivation of large-scale motor neurons (MNs) from human pluripotent stem cells is central to the understanding of MN development,modelling of MN disorders in vitro and development of cell-replacement therapies. Here we develop a method for rapid (20 days) and highly efficient (˜70%) differentiation of mature and functional MNs from human pluripotent stem cells by tightly modulating neural patterning temporally at a previously undefined primitive neural progenitor stage. This method also allows high-yield (textgreater250%) MN production in chemically defined adherent cultures. Furthermore,we show that Islet-1 is essential for formation of mature and functional human MNs,but,unlike its mouse counterpart,does not regulate cell survival or suppress the V2a interneuron fate. Together,our discoveries improve the strategy for MN derivation,advance our understanding of human neural specification and MN development,and provide invaluable tools for human developmental studies,drug discovery and regenerative medicine. View Publication

Serotonergic axons from the raphe nuclei in the brainstem project to every region of the brain,where they make connections through their extensive terminal arborizations. This serotonergic innervation contributes to various normal behaviors and psychiatric disorders. The protocadherin-alpha (Pcdha) family of clustered protocadherins consists of 14 cadherin-related molecules generated from a single gene cluster. We found that the Pcdhas were strongly expressed in the serotonergic neurons. To elucidate their roles,we examined serotonergic fibers in a mouse mutant (Pcdha(Delta CR/Delta CR)) lacking the Pcdha cytoplasmic region-encoding exons,which are common to the gene cluster. In the first week after birth,the distribution pattern of serotonergic fibers in Pcdha(Delta CR/Delta CR) mice was similar to wild-type,but by 3 weeks of age,when the serotonergic axonal termini complete their arborizations,the distribution of the projections was abnormal. In some target regions,notably the globus pallidus and substantia nigra,the normally even distribution of serotonin axonal terminals was,in the mutants,dense at the periphery of each region,but sparse in the center. In the stratum lacunosum-molecular of the hippocampus,the mutants showed denser serotonergic innervation than in wild-type,and in the dentate gyrus of the hippocampus and the caudate-putamen,the innervation was sparser. Together,the abnormalities suggested that Pcdha proteins are important in the late-stage maturation of serotonergic projections. Further examination of alternatively spliced exons encoding the cytoplasmic tail showed that the A-type (but not the B-type) cytoplasmic tail was essential for the normal development of serotonergic projections. View Publication