: Neural stem cells (NSCs) can be propagated in vitro for extensive periods of time while retaining the ability to differentiate into neurons, astrocytes and oligodendrocytes. However, NSCs are limited in their potential to yield specific neuron types. Na?ve NSCs expanded in vitro give rise primarily to GABAergic interneurons and to a lesser extent glutamate neurons. This suggests that NSCs, while multipotent, may not have access to the full spectrum of neuron types. In contrast human embryonic stem cells (hESCs) differentiated towards early neural fates can be readily biased towards various region-specific neuron types such as midbrain dopamine neurons or spinal motoneurons. We have recently reported that hESC derived neural progeny responsive to such regional patterning cues are organized into columnar neuroepithelial structures termed neural rosettes (R- NSCs) and characterized R-NSCs in considerable detail1. Our study has identified R-NSCs as novel, unique NSC stage based on marker expression, clonal stem cell properties, neural differentiation potential, and genetic identity1, 2. The most intriguing finding of these studies was the broad patterning potential of R-NSCs compared to other currently available NSC types. R-NSCs are capable of comprehensive differentiation towards CNS1 and PNS3 derivatives and capable of in vivo engraftment. Despite these exciting preliminary data our studies also revealed major gaps in our current understanding of RNSC biology. Here we would like to address some of these limitations by defining heterogeneity within RNSCs and develop genetic strategies for the prospective isolation of fully patternable R-NSCs based on BAC transgenesis. BAC transgenic hESC reporter lines have been recently pioneered in the Studer lab and will serve as reliable readout of R-NSC stage, identity and function. BAC transgenic reporters will also be critical for probing function of extrinsic and intrinsic factors affecting R-NSC identity. These studies should provide fundamental insights into the genetic and epigenetic mechanisms of neural patterning and ultimately result in novel conditions for the continued in vitro expansion of fully patternable R-NSC - a key step towards establishing a stable expandable universal NSC population.
This proposal defines the molecular factors affecting the fate potential in human ESC derived neural stem cells. The overarching goal is to establish a truly unlimited source of neural stem cells that retains the full capacity of generating any type of neuron in the central and peripheral nervous system. The availability of such highly potent human ESC derived neural stem cell populations should have a major impact for the development of cell therapies in the nervous system and for generating large number of neural cells suitable for drug screening. Our study builds on a set of novel genetic tools developed in the Studer lab BAC transgenesis to precisely define the molecular factors controlling the potency of human neural stem cells in culture. We will use these tools to isolate individual neural stem cell compartments and identify those compartments that exhibit the greatest potency. Based on these molecular markers we will also perform studies aimed at optimizing extrinsic culture conditions for long-term maintenance of the most potent neural stem cell populations. We hope that these studies will ultimately allow us to maintain a universal neural stem cell without any loss of fate potential. Such an embryonic stem cell equivalent of the nervous system would retain the potency of the earliest neural plate stage cells in vitro, in a manner similar to the maintenance of the pluripotent state of the ICM and epiblast in embryonic stem cells. CITED REFERENCES: 1. Elkabetz,Y. et al. Human ES cell-derived neural rosettes reveal a functionally dinstinct early neural stem cell stage. Genes Dev. 22, 152-165 (2008). 2. Elkabetz,Y. &Studer,L. Human ESC-derived Neural Rosettes and Neural Stem Cell Progression. Cold Spring Harb. Symp. Quant. Biol.(2009). 3. Lee,G. et al. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468-1475 (2007). 4. Merkle,F.T., Mirzadeh,Z., &varez-Buylla,A. Mosaic organization of neural stem cells in the adult brain. Science 317, 381-384 (2007). 5. Studer,L., Tabar,V., &McKay,R.D. Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nature Neurosci. 1, 290-295 (1998). 6. Yan,J., Studer,L., &McKay,R.D.G. Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors. J. Neurochem. 76, 307-311 (2001). 7. Perrier,A.L. et al. From the Cover: Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101, 12543-8 (2004). 8. Li,X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215-221 (2005). 9. Lee,H.J. et al. Directed differentiation and transplantation of human embryonic stem cell derived motoneurons. Stem Cells 25, 1931-1939 (2007). 10. Placantonakis,D. et al. BAC transgenesis in human ES cells as a novel tool to define the human neural lineage. Stem Cells 27, 521-532 (2009).
