Isolating Induced Neural Progenitor Cell Colonies Derived From Adult Human Fibroblasts

Samenvatting

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

This video demonstrates a technique for isolating induced neural progenitor cells (iNPCs) derived from adult human fibroblasts. It outlines the steps involved in isolating the iNPC colonies and culturing them in laminin-coated multi-well plate wells in the presence of suitable culture media that facilitate the continued proliferation and expansion of the iNPC population.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Putative Induced Neural Progenitor Cell (iNPC) Colonies

1. One day before picking iNPC colonies, coat one 48 well-plate with laminin: dilute laminin to 1 µg/ml in Dulbecco's Phosphate-Buffered Saline (DPBS), add 300 µl of the solution onto plates, and keep at 4 °C for 24 hr. One day later, aspirate laminin from plates and add 200 µl of neuroinduction media (1:1 DMEM/F-12:Neurobasal, 1x N2, 1x B27, 1% GlutaMAX, 10 ng/ml human leukemia inhibitory factor, 3 μM CHIR9....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
CHIR99021	Axon medchem	1386
DMEM F12	LifeTechnologies	11320-033
DPBS	LifeTechnologies	14190-094
GlutaMAX	LifeTechnologies	35050-038
hLIF	Peprotech	300-05
Laminin	Sigma	L2020
N2	LifeTechnologies	17502-048
Neurobasal	LifeTechnologies	21103-049
SB431542	Invivogen	inh-sb43
Y27632	Calbiochem	CAS 146986-50-7
B27	LifeTechnologies	17504-044