Neural differentiation of induced pluripotent stem cells pdf

CrossRef Google Scholar. Gerrard, L. Karumbayaram, S. Cooper, O. Mol Cell Neurosci. Google Scholar. Vazin, T. Davidson, K. Brain Res. Denham, M. Hotta, R. PubMed Google Scholar. Li, X. Bao-Yang Hu , a Jason P.

Thomson , b, c and Su-Chun Zhang a, c, d, 1. Jason P. James A. Author information Copyright and License information Disclaimer. E-mail: ude. Copyright notice. This article has been cited by other articles in PMC. Abstract For the promise of human induced pluripotent stem cells iPSCs to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages.

Keywords: neural development, regeneration, reprogramming, transplantation, motor neurons. Open in a separate window. Discussion Our present study shows that human iPSCs differentiate to the neural lineage according to the same temporal program as that of hESCs.

Neuron and Glial Differentiation. Immunocytochemistry and Microscopy. Quantification and Statistics. Electrophysiological Recording. Supplementary Material Supporting Information: Click here to view. Footnotes Conflict of interest statement: J.

References 1. Perrier AL, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Li XJ, et al. Specification of motoneurons from human embryonic stem cells.

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Dimos JT, et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Ebert AD, et al. Induced pluripotent stem cells from a spinal muscular atrophy patient. Lee G, et al. Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.

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Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells. Karumbayaram S, et al. Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells. Choi KD, et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells.

Abeyta MJ, et al. Unique gene expression signatures of independently-derived human embryonic stem cell lines. Learn More. Induced Pluripotent Stem Cells iPSCs are pluripotent stem cells that can be generated from somatic cells, and provide a way to model the development of neural tissues in vitro. One particularly interesting application of iPSCs is the development of neurons analogous to those found in the human forebrain.

Here, we present our protocol for differentiating iPSCs into forebrain neural progenitor cells NPCs and neurons, whereby neural rosettes are generated from stem cells without dissociation and NPCs purified from rosettes based on their adhesion, resulting in a more rapid generation of pure NPC cultures.

Neural progenitor cells can be maintained as long-term cultures, or differentiated into forebrain neurons. This protocol provides a simplified and fast methodology of generating forebrain NPCs and neurons, and enables researchers to generate effective in vitro models to study forebrain disease and neurodevelopment.

This protocol can also be easily adapted to generate other neural lineages. Induced pluripotent stem cells iPSCs are stem cells produced from non-pluripotent source cells and tissues Shi et al.

Due to their ability to differentiate into a wide range of cell types, they are a promising avenue for improving our understanding of human development and treatment of degenerative diseases Marchetto et al. Of particular interest are iPSC-derived models of human forebrain neurons, as these cells are known to mediate higher order brain functions, including consciousness Baxter and Chiba, , emotion Morgane et al.

As there are few effective forebrain models for humans, the discovery of iPSCs spurred a rapid push to develop effective protocols to differentiate iPSCs to forebrain neurons Srikanth and Young- Pearse, The first protocols that were developed drew upon previous work using embryonic stem cells ESCs , which relied upon feeder cell cultures.

This complicated the procedure and raised concerns about clinical applications. Later protocols were able to generate forebrain neurons without using feeder cells Bell et al. It can be difficult to make an all-encompassing statement about the protocols currently used to generate forebrain NPCs, due to the multitude of labs currently generating forebrain neurons and the many variables that can be changed and optimized. However, many of the recently most cited published protocols for the generation of forebrain NPCs and neurons can be divided into two kinds, monolayer and embryoid bodies EBs protocols.

In an EB based protocol, iPSCs are dissociated and plated in suspension in a neural induction media to allow them to form EBs, which gradually aggregate over days Pasca et al. These EBs are then transferred to a plate that supports cell attachment, enabling the embryoid bodies to attach to the bottom of the plate and spread out into a neural rosette.

From this rosette, neural stem cells NSCs arise, which can be passaged to form relatively stable neural progenitors cells NPCs Shi et al. NPCs can then be plated in a neuronal induction media to give rise to mature neurons Bell et al. Monolayer based protocols chiefly differ in that iPSC colonies are maintained as a monolayer during neural induction, and develop directly into rosettes without aggregation Chandrasekaran et al.

This protocol describes a methodology for generating forebrain neurons from iPSCs, where iPSC colonies are induced to form neural rosettes without mechanical dissociation, and neural progenitor cells are purified from immature clusters of neural cells, known as neural rosettes based on differential adhesion. Neural progenitor cells will not attach to non-adherent plates and aggregate together in a floating mass, while other cells types either adhere or float but do not aggregate with NPCs Bell et al.

This allows rapid purification of NPCs, has the potential for automation and enables the generation of NPC cultures within 14 days of initiation of differentiation. This modification does not appear to negatively influence the fate of the cells, as we observe uniform staining for key neural progenitor cells markers Zhang et al. Indeed, we have found that we are capable of recording electrical activity from neurons consistently in as little as five days of differentiation from NPCs.

This protocol can be used to generate forebrain neurons simply and effectively for use in investigating neurodevelopment, the etiology of diseases that affect the forebrain, and drug testing. Clampex GraphPad Prism 7 GraphPad, www. In order for differentiation to proceed effectively, ensure that you begin differentiation with high-quality iPSC cultures.

IPSCs should be free of karyotypic abnormalities C , possess the ability to differentiate into all three germ lineages and express characteristic markers of each lineage D , and test negative for mycoplasma contamination E. Working in a class II biological safety cabinet, use appropriately sized pipettes to plate iPSCs in E8 medium in a mm dish. If starting from a frozen aliquot of iPSC, we recommend plating at least , cells. This is considered Day 0 of iPSC culture.

Add 3 ml of complete Neural Induction Medium 1, pre-warmed in a bead bath to each plate using a 5 ml pipette. If plates do not contain colonies of sufficient size, add 3 ml of E8 media and check daily until colonies reach the appropriate size. On Day 2 about 48 h after switching to Neural Induction Medium , change the medium by aspirating old medium from each well. Add 3 ml of pre-warmed complete Neural Induction Medium 1 to each plate. On Day 4 of neural induction, cells will be reaching confluency.

If necessary , mark any colonies with non-neural differentiation. Aspirate the spent medium from each well. Note: Due to high cell density in the culture from Day 4 onwards, doubling the volume of Neural Induction Medium is very critical for cell nutrition.

Also, minimal cell death should be observed from Days 4 to 7 after neural induction. If the color of cells turns yellowish with many floating cells during Days 4 to 7 of neural induction, it indicates that the starting density of iPSCs was too high.

Ideally, work with these variables to ensure that the media does not continue to turn yellow. On Day 6 of neural induction, cells should be near maximal confluence. Remove any non-neural differentiated cells that can be observed and add 3 ml of complete Neural Induction Medium into each plate. On Day 7 of neural induction, the medium should be switched into Neural Induction Medium 2. Add 3 ml of complete Neural Induction Medium 2 to each plate. The medium should be changed every day for 5 days.

For example morphologies, see Figure 2. Day 2: The iPSC colony, which has been treated with Neural Induction Media 1 for 2 days, begins to change cellular morphology and some cells extend processes. Day 5: Increased expansion of the colony with some differentiation of outer cells. Day Appearance of rosettes in the colony become visible. NSCs are present in high confluence in the middle of these structures. It is at this point that colonies are detached and re-plated on non-adherent plates at D13 for two days.

D13 immediately after plating on adherent plates. This image shows floating rosette colonies that will continue to proliferate and differentiate in a floating mass. Non-rosette cells either remained on the dish at D12 after chemical release or float as single cells on the non-adherent plates shown in E. At D15, rosette clusters expand in size and are moved to adherent plates. Cell aggregates here are 3-dimensional, but are attached to the plate.

Note the purity of the clusters at this point F. Add 1. Tap plates gently to dislodge cells still attached. Use a pipette to gently rinse the surface of the plates with the Gentle Cell Dissociation Reagent already in the plates to detach any remaining cells.

Add 1 ml of DPBS to each plate to collect residual cells and transfer the cell suspension to the conical tube. Gently pipet the cell suspension up and down 3 times with a 5-ml or ml pipette to break up the cell clumps. Culture the cells in a CO 2 incubator for 2 days.