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Directed differentiation of human embryonic stem cells (hESCs) to a functional cell type, including neurons, is the foundation for application of hESCs. We describe here a reproducible, chemically-defined protocol that allows directed differentiation of hESCs to nearly pure neuroectodermal cells and neurons. First, hESC colonies are detached from mouse fibroblast feeder layers and form aggregates to initiate the differentiation procedure. Second, after 4 days of suspension culture, the ESC growth medium is replaced with neural induction medium to guide neuroectodermal specification. Third, the differentiating hESC aggregates are attached onto the culture surface at day 6-7, where columnar neural epithelial cells appear and organize into rosettes. Fourth, the neural rosettes are enriched by detaching rosettes and leaving the peripheral flat cells attached, and expanded as neuroepithelial aggregates in the same medium. Finally, the neuroepithelial aggregates are dissociated and differentiated to nearly pure neurons. This stepwise differentiation protocol results in the generation of primitive neuroepithelia at day 8-10, neural progenitors at the 2nd and 3rd week, and postmitotic neurons at the 4th week, which mirrors the early phase of neural development in a human embryo. Identification of the primitive neuroepithelial cells permits efficient patterning of region-specific progenitors and neuronal subtypes such as midbrain dopaminergic neurons.
Directed differentiation of specific lineages has been a focal point in the field of human embryonic stem cell (hESC) research (1, 2). The differentiation paradigms are key to revealing the cellular and molecular mechanisms underlying early human development, exploring tissue engineering, and devising cell replacement therapy and drug screening. Ideally, the differentiation protocol should be based on the developmental principles and truly directed (controllable). It should also be simple, defined, and reproducible.
We have developed a reproducible, chemically-defined protocol that allows directed differentiation of hESCs to a synchronized population of neuroectodermal cells and then a nearly pure population of neurons (3, 4). The design of the protocol rests essentially on the fundamental principle of neuroectodermal induction (5). As such, the specification of neuroepithelial cells and subsequent neuronal and glial differentiation mirrors the time course of human embryo development predicted from animal embryological studies and limited human specimens. We avoid any unknown components, such as sera or stroma cells, in the system so that the culture is chemically-defined, allowing flexible modification for mechanistic analyses. Because of the simplicity, the protocol has been proven highly reproducible for the past decade. The neural differentiation is also robust with over 90% efficiency (3, 4). This endows the system particularly amenable for biochemical analysis that often requires materials of large amount. Technically, the adherent colony culture also permits continual observation of morphological transformation along the differentiation processes.
Under this protocol, differentiating cells manifest five morphologically identifiable stages, the ESCs, ESC aggregates, neuroepithelia in the form of neural tube-like rosettes, neural progenitors in neuroepithelial aggregates, and postmitotic neurons. The undifferentiated state of the starting cell population is essential for the successful replication of the protocol. The quality of hESCs is often overlooked when it comes to differentiation. In reality, partially differentiated hESCs will unavoidably result in unsynchronized differentiation, such as the generation of postmitotic neurons within the first week along with neuroepithelial generation. Human ESCs, cultured on mouse embryonic fibroblast (MEF) feeder layers, are usually passaged every 5-6 days before differentiation. At this stage, the ESC colonies are generally uniform in size, and they form aggregates of similar size after detaching from the MEF layers. These ESC aggregates are initially suspended in the ESC growth medium for 4 days to promote cell survival and then the medium is replaced with the serum-free neural induction medium to guide the cells toward the ectoderm fate. This is evidenced by transient expression of FGF5 and uniform expression of Otx2 and Sox2 but lack of brachyury and α-fetoprotein (AFP), meso-endodermal markers (4). Prolonged culture in the ESC growth medium will lead to the formation of “embryoid bodies”, which is not ideal for neural differentiation.
Unlike most of the neural differentiation protocols that involve long-term aggregated culture or co-culture with stroma cells, in our protocol, the ESC aggregates are reseeded onto a culture dish (without feeder) and they reform individual monolayer colonies. This permits individual cells to be exposed to the culture environment evenly for synchronized differentiation. It also allows morphological observation. After 8-10 days of differentiation, neuroepithelia, characterized by columnar epithelial morphology, appear. Their identity is confirmed by expression of a host of neuroectodermal transcription factors including Pax6, Lhx2, Six3, and Sox2, and the lack of other germ layer markers (4). By two weeks of differentiation, these epithelial cells organize into rosettes, reminiscent of the cross section of the neural tube. These cells express Sox1 in addition to the above neural markers and exhibit polarity by concentrated expression of N-cadherin in the lumen (3, 4). This stage of differentiation can be subdivided into two morphologically distinct stages. The early columnar neuroepithelia, which we term primitive anterior neuroepithelia, uniformly express anterior transcription factors but can be readily specified to other regional identities (6-9). Therefore, the primitive neuroepithelia behave like true neural stem cells. We will use midbrain dopamine neuron differentiation to illustrate the point. The late columnar neuroepithelia, usually with a lumen inside and expression of definitive neural marker Sox1, can not be patterned to region-specific progenitors and are termed as definitive neuroepithelia (7, 9).
The neuroepithelia in the form of neural tube-like rosettes can be readily detached from the culture surface and expanded in suspension like “neurospheres” although these cells are relatively primitive comparing to the progenitor aggregates derived from the embryonic or adult brain. These cells, upon dissociation and plating onto substrate, will differentiate to postmitotic neurons.
This study was supported by the National Institute of Neurological Disorders and Stroke (NS045926, NS046587), the Michael J. Fox Foundation, and partly by a core grant to the Waisman Center from the National Institute of Child Health and Human Development (P30 HD03352).
1If filtration is needed, we recommend using disposable receipt bottle (Stericup, Millipore) other than reused glass bottle.
2The undifferentiated state of ESCs is very important for efficient neural induction. If there are differentiated cells in the ESC culture, floating ESC aggregates are easy to attach to the flask and neurons will be easily found in the culture dish when these aggregates are plated down (see Fig1 H and I)
3Usually the size of the clusters is around twice the size for ESCs for passaging/splitting. Cells will die and the yield of ESC aggregates will be quite low if the size of the clusters is too small. The differentiation will be unsynchronized if the size is too large.
4As an alternative to the attachment of ESC aggregates using laminin, FBS can also be used. Add neural induction medium containing 10% FBS to the ESC aggregates as mentioned in subheading 3.2. Plate 1-1.5 ml of medium containing 30-40 aggregates to each well of a 6-well plate and let the cells attach in the incubator overnight. Right after the cells attach, wash the cells with warm DMEM/F12 once and feed with neural induction medium. Please note that it is better to leave the cells in the presence of serum for as short time as possible.
5Coverslips need be pretreated with nitric acid for 1 hour and then 95% ethanol for 30 min after washing with distilled water. These coverslips then need be coated with 0.1 mg/ml polyornithine overnight before use (for details see www.wicell.org).
6Flame polish a cotton plugged Pasteur pipette to narrow the opening end slightly and make a 20-30° bend at the narrow part of the shaft. Pipette up and down 1-3 times to let the aggregates go through the bended pipette and shear them into small pieces. In this way, neuroepithelial aggregates can be expanded from 1 flask into 2.
7Cells can be dissociated to either single cells or small clusters. Small clusters tend to survive better than single cells. For dopaminergic differentiation, we dissociate the cells to single cells and plate at a high density.
8L cells that express Wnt3a (ATCC, Manassas, VA) are conditioned in neural differentiation medium for 24 hours and the conditioned medium is used at a final concentration of 50% from day 24 to day 44 to promote the proliferation of specified DA progenitors.