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Generating regionalized neuronal cells from pluripotency, a step-by-step protocol.

Kirkeby A, Nelander J, Parmar M - Front Cell Neurosci (2013)

Bottom Line: Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling.Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion.Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science and Lund Stem Cell Center, Lund University Lund, Sweden.

ABSTRACT
Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or in vitro analysis.

No MeSH data available.


Related in: MedlinePlus

Overview of differentiation protocol. Cells are differentiated as EBs for the first 4 days, and then cultured as attached colonies on coated plates with subsequent dissociation and replating at day 11. Neuralisation and patterning factors are present between day 0–9 (only SB431542 is withdrawn at day 7), and terminal differentiation is initiated from day 14 and onward with db-cAMP and DAPT.
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Figure 1: Overview of differentiation protocol. Cells are differentiated as EBs for the first 4 days, and then cultured as attached colonies on coated plates with subsequent dissociation and replating at day 11. Neuralisation and patterning factors are present between day 0–9 (only SB431542 is withdrawn at day 7), and terminal differentiation is initiated from day 14 and onward with db-cAMP and DAPT.

Mentions: The protocol presented here will produce neural cells of a telencephalic fate in the absence of any patterning factors. To control rostro-caudal and dorso-ventral patterning, see separate section below. This protocol is preferably started on a Monday to avoid medium changes during the weekends (see overview of differentiation procedure in Figure 1). It is important to start the differentiation with healthy, pluripotent cells. All differentiated colonies should be removed from the culture before initiating differentiation.


Generating regionalized neuronal cells from pluripotency, a step-by-step protocol.

Kirkeby A, Nelander J, Parmar M - Front Cell Neurosci (2013)

Overview of differentiation protocol. Cells are differentiated as EBs for the first 4 days, and then cultured as attached colonies on coated plates with subsequent dissociation and replating at day 11. Neuralisation and patterning factors are present between day 0–9 (only SB431542 is withdrawn at day 7), and terminal differentiation is initiated from day 14 and onward with db-cAMP and DAPT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539732&req=5

Figure 1: Overview of differentiation protocol. Cells are differentiated as EBs for the first 4 days, and then cultured as attached colonies on coated plates with subsequent dissociation and replating at day 11. Neuralisation and patterning factors are present between day 0–9 (only SB431542 is withdrawn at day 7), and terminal differentiation is initiated from day 14 and onward with db-cAMP and DAPT.
Mentions: The protocol presented here will produce neural cells of a telencephalic fate in the absence of any patterning factors. To control rostro-caudal and dorso-ventral patterning, see separate section below. This protocol is preferably started on a Monday to avoid medium changes during the weekends (see overview of differentiation procedure in Figure 1). It is important to start the differentiation with healthy, pluripotent cells. All differentiated colonies should be removed from the culture before initiating differentiation.

Bottom Line: Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling.Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion.Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science and Lund Stem Cell Center, Lund University Lund, Sweden.

ABSTRACT
Human pluripotent stem cells possess the potential to generate cells for regenerative therapies in patients with neurodegenerative diseases, and constitute an excellent cell source for studying human neural development and disease modeling. Protocols for neural differentiation of human pluripotent stem cells have undergone significant progress during recent years, allowing for rapid and synchronized neural conversion. Differentiation procedures can further be combined with accurate and efficient positional patterning to yield regionalized neural progenitors and subtype-specific neurons corresponding to different parts of the developing human brain. Here, we present a step-by-step protocol for neuralization and regionalization of human pluripotent cells for transplantation studies or in vitro analysis.

No MeSH data available.


Related in: MedlinePlus