Differentiating Induced Neural Progenitor Cells into Astrocytes

Samenvatting

Source: Meyer, S., et al., Edenhofer, F. Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells. J. Vis. Exp. (2015)

This video demonstrates the process for differentiating induced neural progenitor cells (iNPCs) into neurons and astrocytes using specialized media.

Protocol

1. Differentiation of iNPCs

1. Differentiation towards neuronal lineage​
   1. Culture cells on laminin-coated plates as described above. Change media to neuro-diff-media (DMEM/F-12, 1x N2, 1x B27, 300 ng/ml cAMP, 200 µM vitamin C, 10 ng/ml BDNF, 10 ng/ml GDNF) when cells are approximately 70% confluent. Change media every other day for three weeks. Do not split cells during differentiation.
   2. After three weeks, cells should express the neuronal marker TUJ1. To gain more mature neurons and neuronal subtypes, continue differentiation upto 3 months.
2. Differentiation towards glial lineage
   1. Change media to glial induction media (1:1 DMEM/F-12 : Neurobasal, 1x N2, 1x B27, 10 µM β-mercaptoethanol, 100 µM non-essential amino acids, 1.5 mM L-Glutamine, 5 µg/ml insulin, 40 ng/ml T3) including 20 ng/ml EGF to induce differentiation into glial precursor cells. Remove half of the media and replace with fresh media every other day for about 2 weeks. During this period, cells should be split 1:3 in the presence of 10 µM ROCK inhibitor Y27632 when 100% confluency is reached.
   2. After 2 weeks, differentiate cells further into astrocytes: when cells are nearly confluent, change media to commercially available astrocyte media, change media every second day. After about 7 days, cells start to express astrocyte markers (e.g., GFAP). If cells get 100% confluent during the differentiation protocol, split them in a 1:2 ratio in the presence of 10 µM ROCK inhibitor Y27632.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Astrocyte media	ScienCell	1801
B27	LifeTechnologies	17504-044
BDNF	Peprotech	450-02
cAMP	Sigma	A6885
DMEM	LifeTechnologies	41966-029
DMEM F12	LifeTechnologies	11320-033
DMSO	Roth	4720
DPBS	LifeTechnologies	14190-094
EGF	Life Technologies	PHG0313
FCS	Biochrom AG	50115
GDNF	Peprotech	450-10
Insulin	Seralab	GEM-700-112-P
L-Glutamine	LifeTechnologies	25030-024
Laminin	Sigma	L2020
N2	LifeTechnologies	17502-048
Neurobasal	LifeTechnologies	21103-049
Non-essential amino acids	LifeTechnologies	11140-050
Vitamin C	Sigma	A4544
β-Mercaptoethanol	LifeTechnologies	21985023