Direct Conversion Of Mouse Somatic Cells Into Multipotent Neural Stem/Progenitor Cells And Mature Dopaminergic Neurons By Defined Factors

Neurodegenerative disorders have already become one of the most serious diseases that affect human health. Parkinson’s disease (PD), for example, is a neurodegenerative disease that afflicts about1%of the population over age65around the world. One of the pathological hallmarks of PD is the degeneration of dopaminergic (DA) neurons at midbrain. The relatively focal lesion feature of PD and the limited regenerative ability of nervous system make cell replacement therapy a promising approach for treating this disease. Embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent unlimited sources of donor cells for cell therapy. However, the risk of tumor formation resulting from even a very small number of contaminating pluripotent cells in the graft and the relatively low efficiency of DA neuron generation still remain the major caveats for the clinical applications of ES and iPS cells-derived cells. Somatic-derived directly converted mature neurons (induced neurons, iNs) provide an alternative strategy for cell transplantation therapies, nevertheless iNs are terminally differentiated cells that have completely exited cell cycle and thus are not capable of proliferation and would have difficulty surviving transplantation. Therefore, self-renewing, multipotent neural stem/progenitor cells (NSC/NPC) capable of DA neuron specification will hold great promise for treating Parkinson’s disease.In the present study, we have successfully converted different mouse somatic cells into a specific neuronal subtype-dopaminergic neurons through two separate routes. One is to first induce somatic cells into neural progenitor cells and then differentiate them to mature DA neurons; and the other is to directly convert somatic cells into functional DA neurons with DA lineage specific factors combined with the two iN factors, Ascll and Brn2. The results are listed below:(1) Sertoli cells are mesoderm-originated terminally differentiated cells that provide a nurturing microenvironment for spermatogenesis. Sertoli cell can be cultured at high purity to avoid the heterogeneity problems inherent in mouse dermal fibroblast cultures, thus may serve as a wonderful model to test the principles of trans-germ-layer differentiation and somatic-to-multipotent conversion. We demonstrate that sertoli cells can be directly converted into a multipotent state that possesses neural stem/progenitor cell properties by a set of exogenous factors (Ascl1, Brn2, Ngn2, Hes1, Id1, Pax6, Sox2, c-Myc, and Klf4). The sertoli cell-derived induced neural stem/progenitor cells (SC-iNPCs) express multiple NSC-specific markers, exhibit a global gene-expression profile similar to normal NSCs, and are capable of self-renewal and differentiating into glia and electrophysiologically functional neurons. SC-iNPC-derived neurons stain positive for tyrosine hydroxylase (TH), y-aminobutyric acid (GABA), and choline acetyltransferase (ChAT). In addition, SC-iNPCs can survive, give rise to mature neurons, and generate synapses following transplantation into the adult mouse dentate gyrus.(2) Dermal fibroblasts are considered as important source materials for regenerative medicine, since they can be easily obtained in clinic. Here we chose postnatal mouse tail-tip fibroblasts (TTFs) as the originating cells. By introduction of the same set of exogenous factors used for sertoli cell conversion, TTFs can also be directly induced into multipotent neural stem/progenitor cells (TTF-iNPCs), which display very similar characteristics with brain-derived normal NSCs and SC-iNPCs, and are also capable of mature DA neuron differentiation.(3) Mouse embryonic fibroblasts (MEFs) can be directly converted into mature neurons by only two iN factors-Ascll and Brn2. Furthermore, we find that different groups of DA lineage specific factors (such as Nurrl, Lmxlb and Otx2, or Foxa2and Lmxla) combined with Ascll and Brn2can directly induce MEFs into mature midbrain DA neurons.The successful direct conversion of mulitipotent iNPCs and mature DA neurons from mesoderm-derived somatic cells may provide bran-new platforms and cell resources for futher regenerative medicine. Furthermore, my studies unambiguously demonstrate that terminally differentiated somatic cells can be directly converted to other cell types from different germ layers and at different developmental status.