Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.     

Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences

The mechanisms governing the emergence of the earliest mammalian neural cells during development remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition; however, an instructive process has also been proposed. We used mouse embryonic stem (ES) cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals and what cellular fate will result. To assess this default state, ES cells were placed in conditions that minimize external influences. Individual ES cells were found to rapidly transition directly into neural cells, a process shown to be independent of suggested instructive factors (e.g., fibroblast growth factors). Further, we provide evidence that the default neural identity is that of a primitive neural stem cell (NSC). The exiguous conditions used to reveal the default state were found to present primitive NSCs with a survival challenge (limiting their persistence and proliferation), which could be mitigated by survival factors or genetic interference with apoptosis.     

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

Generation of a defined and uniform population of CNS progenitors and neurons from mouse embryonic stem cells

A detailed protocol is described allowing the generation of essentially pure populations of glutamatergic neurons from mouse embryonic stem (ES) cells. It is based on the culture of ES cells that are kept undifferentiated by repeated splitting and subsequently amplified as non-adherent cell aggregates. Treatment with retinoic acid causes these ES cells to essentially become neural progenitors with the characteristics of Pax6-positive radial glial cells. As they do in vivo, these progenitors differentiate in glutamatergic pyramidal neurons that form functional synaptic contacts and can be kept in culture for long periods of time. This protocol does not require the use of ES lines expressing resistance or fluorescent markers and can thus be applied in principle to any wild-type or mutant ES line of interest. At least 2 weeks are required from starting ES cell culture until plating progenitors and differentiating neurons establish synaptic transmission within about 10 days.     

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.     

Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca²⁺ mobilizing nucleotide presented in various species. NAADP mobilizes Ca²⁺ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.     

Mature endothelium and neurons are simultaneously derived from embryonic stem cells by 2D in vitro culture system

The connections existing between vessels and nerves go beyond the structural architecture of vascular and nervous systems to comprise cell fate determination. The analysis of functional/molecular links that interconnect endothelial and neural commitments requires a model in which the two differentiation programs take place at the same time in an artificial controllable environment. To this regard, this work presents an in vitro model to differentiate embryonic stem (ES) cells simultaneously into mature neurons and endothelial cells. Murine ES cells are differentiated within an artificial environment composed of PA6 stromal cells and a serum-free medium. Upon these basal culture conditions ES cells preferentially differentiate into neurons. The addition of basic fibroblast growth factor (FGF2) to the medium allows the simultaneous maturation of neurons and endothelial cells, whereas bone morphogenetic protein (BMP)4 drives endothelial differentiation to the disadvantage of neural commitment. The responsiveness of the system to exogenous cytokines was confirmed by genes expression analysis that revealed a significant up-regulation of endothelial genes in presence of FGF2 and a massive down-regulation of the neural markers in response to BMP4. Furthermore, the role played by single genes in determining endothelial and neural fate can be easily explored by knocking down the expression of the target gene with lentiviruses carrying the corresponding shRNA sequence. The possibility to address the neural and the endothelial fate separately or simultaneously by exogenous stimuli combined with an efficient gene silencing strategy make this model an optimal tool to identify environmental signals and genes pathways involved in both endothelial and neural specification.     

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.     

Mu- and kappa-opioids induce the differentiation of embryonic stem cells to neural progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected mu-opioid receptor (MOR-1) and kappa-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [D-Ala(2),MePhe(4),Gly-ol(5)]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.     

Neuronal differentiation of cryopreserved neural progenitor cells derived from mouse embryonic stem cells

Embryonic stem cells (ES cells) are developmentally pluripotent cells isolated from pre-implantation mammalian embryos. In cell culture ES cells can be easily differentiated to generate cultures of neural progenitors. We present a simple method for the cryopreservation of these ES-derived neural progenitors. Cryopreserved neural progenitor stocks can be thawed, expanded with FGF2, and differentiated into functional neurons. This method will facilitate studies using ES-derived neural progenitor cells as a cell culture model system for neural development and differentiation. It will also aid studies designed to test the ability of these progenitor cells to functionally engraft and repair damaged neural tissue.     

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors

Elucidating the in vitro differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.     

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.     

Region-specific generation of functional neurons from naive embryonic stem cells in adult brain

Embryonic stem (ES) cells are multipotent progenitors with unlimited developmental potential, and in vitro differentiated ES cell-derived neuronal progenitors can develop into functional neurons when transplanted in the central nervous system. As the capacity of naive primary ES cells to integrate in the adult brain and the role of host neural tissue therein are yet largely unknown, we grafted low densities of undifferentiated mouse ES (mES) cells in adult mouse brain regions associated with neurodegenerative disorders; and we demonstrate that ES cell-derived neurons undergo gradual integration in recipient tissue and acquire morphological and electrophysiological properties indistinguishable from those of host neurons. Only some brain areas permitted survival of mES-derived neural progenitors and formed instructive environments for neuronal differentiation and functional integration of naive mES cells. Hence, region-specific presence of microenvironmental cues and their pivotal involvement in controlling ES cell integration in adult brain stress the importance of recipient tissue characteristics in formulating cell replacement strategies for neurodegenerative disorders.     

Function of Mouse Embryonic Stem Cell-Derived Supporting Cells in Neural Progenitor Cell Maturation and Long Term Cxpansion

Background

In the differentiation of mouse embryonic stem (ES) cells into neurons using the 5-stage method, cells in stage 4 are in general used as neural progenitors (NPs) because of their ability to give rise to neurons. The choice of stage 4 raises several questions about neural progenitors such as the type of cell types that are specifically considered to be neural progenitors, the exact time when these progenitors become capable of neurogenesis and whether neurogenesis is an independent and autonomous process or the result of an interaction between NP cells and the surrounding cells.

Methodology/Principal Findings

In this study, we found that the confluent monolayer cells and neural sphere like cell clusters both appeared in the culture of the first 14 days and the subsequent 6 weeks. However, only the sphere cells are neural progenitors that give rise to neurons and astrocytes. The NP cells require 14 days to mature into neural lineages fully capable of differentiation. We also found that although the confluent monolayer cells do not undergo neurogenesis, they play a crucial role in the growth, differentiation, and apoptosis of the sphere cells, during the first 14 days and long term culture, by secreted factors and direct cell to cell contact.

Conclusions/Significance

The sphere cells in stage 4 are more committed to developing into neural progenitors than monolayer cells. Interaction between the monolayer cells and sphere cells is important in the development of stage 4 cell characteristics.