CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells.

Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.

NPTX1 regulates neural lineage specification from human pluripotent stem cells.

Neural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood. Here we show that NPTX1, a secreted protein, is rapidly upregulated during neural induction from human pluripotent stem cells (hPSCs). By manipulating its expression, we were able to reduce or initiate neural lineage commitment. A time-course transcriptome analysis and functional assays show that NPTX1 acts in part by binding the Nodal receptor cofactor TDGF1, reducing both Nodal and BMP signaling. Our findings identify one of the earliest genes expressed upon neural induction and provide insight into human neural lineage specification.

Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding.

An essential aspect of stem cell culture is the successful maintenance of the undifferentiated state. Many types of stem cells are FGF2 dependent, and pluripotent stem cells are maintained by replacing FGF2-containing media daily, while tissue-specific stem cells are typically fed every 3rd day. Frequent feeding, however, results in significant variation in growth factor levels due to FGF2 instability, which limits effective maintenance due to spontaneous differentiation. We report that stabilization of FGF2 levels using controlled release PLGA microspheres improves expression of stem cell markers, increases stem cell numbers and decreases spontaneous differentiation. The controlled release FGF2 additive reduces the frequency of media changes needed to maintain stem cell cultures, so that human embryonic stem cells and induced pluripotent stem cells can be maintained successfully with biweekly feedings.

Efficient derivation of functional floor plate tissue from human embryonic stem cells.

The floor plate (FP) is a critical signaling center during neural development located along the ventral midline of the embryo. Little is known about human FP development because of the lack of tissue accessibility. Here we report the efficient derivation of human embryonic stem cell (hESC)-derived FP tissue capable of secreting Netrin-1 and SHH and patterning primary and hESC derived tissues. FP induction in hESCs is dependent on early SHH exposure and occurs at the expense of anterior neurectoderm (AN). Global gene expression and functional studies identify SHH-mediated inhibition of Dkk-1 as key factor in FP versus AN specification. hESC-derived FP tissue is shown to be of anterior SIX6+ character but is responsive to caudalizing factors suppressing SIX6 expression and inducing a shift in usage of region-specific SHH enhancers. These data define the early signals that drive human FP versus AN specification and determine regional identity in hESC-derived FP.

Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.

The isolation of human induced pluripotent stem cells (iPSCs) offers a new strategy for modelling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy, caused by a point mutation in the IKBKAP gene involved in transcriptional elongation. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood owing to the lack of an appropriate model system. Here we report the derivation of patient-specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC-derived lineages demonstrates tissue-specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript, suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell-based assays revealing marked defects in neurogenic differentiation and migration behaviour. Furthermore, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining new insights into human disease pathogenesis and treatment.

Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.

Bmi-1 cooperates with Foxg1 to maintain neural stem cell self-renewal in the forebrain.

Neural stem cells (NSCs) persist throughout life in two forebrain areas: the subventricular zone (SVZ) and the hippocampus. Why forebrain NSCs self-renew more extensively than those from other regions remains unclear. Prior studies have shown that the polycomb factor Bmi-1 is necessary for NSC self-renewal and that it represses the cell cycle inhibitors p16, p19, and p21. Here we show that overexpression of Bmi-1 enhances self-renewal of forebrain NSCs significantly more than those derived from spinal cord, demonstrating a regional difference in responsiveness. We show that forebrain NSCs require the forebrain-specific transcription factor Foxg1 for Bmi-1-dependent self-renewal, and that repression of p21 is a focus of this interaction. Bmi-1 enhancement of NSC self-renewal is significantly greater with increasing age and passage. Importantly, when Bmi-1 is overexpressed in cultured adult forebrain NSCs, they expand dramatically and continue to make neurons even after multiple passages, when control NSCs have become restricted to glial differentiation. Together these findings demonstrate the importance of Bmi-1 and Foxg1 cooperation to maintenance of NSC multipotency and self-renewal, and establish a useful method for generating abundant forebrain neurons ex vivo, outside the neurogenic niche.

High-throughput screening of gene function in stem cells using clonal microarrays.

We describe a microarray-based approach for the high-throughput screening of gene function in stem cells and demonstrate the potential of this method by growing and isolating clonal populations of both adult and embryonic neural stem cells. Clonal microarrays are constructed by seeding a population of cells at clonal density on micropatterned surfaces generated using soft lithographic microfabrication techniques. Clones of interest can be isolated after assaying in parallel for various cellular processes and functions, including proliferation, signal transduction, and differentiation. We demonstrate the compatibility of the technique with both gain- and loss-of-function studies using cell populations infected with cDNA libraries or DNA constructs that induce RNA interference. The infection of cells with a library prior to seeding and the compact but isolated growth of clonal cell populations will facilitate the screening of large libraries in a wide variety of mammalian cells, including those that are difficult to transfect by conventional methods.

shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development.

Knockout studies have shown that the polycomb gene Bmi-1 is important for postnatal, but not embryonic, neural stem cell (NSC) self-renewal and have identified the cell-cycle inhibitors p16/p19 as molecular targets. Here, using lentiviral-delivered shRNAs in vitro and in vivo, we determined that Bmi-1 is also important for NSC self-renewal in the embryo. We found that neural progenitors depend increasingly on Bmi-1 for proliferation as development proceeds from embryonic through adult stages. Acute shRNA-mediated Bmi-1 reduction causes defects in embryonic and adult NSC proliferation and self-renewal that, unexpectedly, are mediated by a different cell-cycle inhibitor, p21. Gene array analyses revealed developmental differences in Bmi-1-controlled expression of genes in the p21-Rb cell cycle regulatory pathway. Our data therefore implicate p21 as an important Bmi-1 target in NSCs, potentially with stage-related differences. Understanding stage-related mechanisms underlying NSC self-renewal has important implications for development of stem cell-based therapies.

The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells.

In the developing cerebral cortex, neurons are born on a predictable schedule. Here we show in mice that the essential timing mechanism is programmed within individual progenitor cells, and its expression depends solely on cell-intrinsic and environmental factors generated within the clonal lineage. Multipotent progenitor cells undergo repeated asymmetric divisions, sequentially generating neurons in their normal in vivo order: first preplate cells, including Cajal-Retzius neurons, then deep and finally superficial cortical plate neurons. As each cortical layer arises, stem cells and neuroblasts become restricted from generating earlier-born neuron types. Growth as neurospheres or in co-culture with younger cells did not restore their plasticity. Using short-hairpin RNA (shRNA) to reduce Foxg1 expression reset the timing of mid- but not late-gestation progenitors, allowing them to remake preplate neurons and then cortical-plate neurons. Our data demonstrate that neural stem cells change neuropotency during development and have a window of plasticity when restrictions can be reversed.