Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Summary

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Abstract

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Introduction

Human embryonic stem cells (hESCs) have the potential to differentiate into the three primary germ layers, which then differentiate into various multipotent progenitor cell lineages. These lineages subsequently give rise to all cell types in the human body. In vitro hESC culture systems have transformed the ability to study human embryonic development and have served as a valuable tool for obtaining new insights into how these processes are regulated at the biological and molecular levels. Similarly, studies of induced pluripotent stem cells (iPSCs) generated from reprogramming somatic cells isolated from human patients provide novel insights into various diseases. In....

Protocol

1. Preparation of hESC-qualified Basement Membrane Matrix-coated Plates

1. Slowly thaw the hESC-qualified basement membrane matrix (see Table of Materials) solution at 4 °C for at least 2–3 h or overnight to avoid formation of a gel.
2. To prepare basement membrane matrix-coated plates, dilute matrix in cold DMEM/F12 to a 2% final concentration. Mix well and coat each well of a 6 well plate with 1 mL of the diluted matrix solution.
3. Incubate the basement membrane matri.......

Discussion

Scalable and efficient methods for the differentiation of hESCs into various lineages and the generation of sufficient numbers of differentiated cells are important criteria for drug screening and stem cell therapy. Various single-cell passing methods have been published, in which cells are cultured in the presence of ROCK inhibitor or other small molecules to improve survival, but the final products of these culture methods are colony type hESCs^17,18,.......

Materials

Name	Company	Catalog Number	Comments
35 mm m-dishes	ibidi	81156	Cell culture dish
6-well plates	Corning	3516
Accutase	Innovative Cell Technologies	AT104-500	Cell detachment solution
Activin A	R&D system	338-AC-050
Ascorbic Acid	Sigma Aldrich	A4403
B27 supplement	Thermo Fisher	17504044
B27 supplement (-Vit A)	Thermo Fisher	12587010
BDNF	Applied Biological Materials	Z100065
bFGF	Peprotech	100-18C
Centrifuge	DAMON/ICE	428-6759
CO2 incubator	Thermo Fisher	4110
Corning hESC-qulified Matrix (Magrigel)	Corning	354277	Basement membrane matrix (used for most of the protocol here)
Cryostor CS 10	Stemcell Technologies	7930	Cell freezing solution
Dispase	Stemcell Technologies	7923
DMEM	Thermo Fisher	10569-010
DMEM/F12	Thermo Fisher	10565-018
Dorsomorphin	Tocris	3093
EGF	Peprotech	AF-100-16A
Fetal Bovine Serum	Fisher Scientific	SH3007003HI
FGF8	Applied Biological Materials	Z101705
GDNF	Applied Biological Materials	Z101057
Geltrex matrix	Thermo Fisher	A1569601	Basement membrane matrix
GlutaMax	Thermo Fisher	35050061	Glutamine supplement, 100X
H9 (WA09) human embryonic stem cell line	WiCell	WA09
Heregulin b-1	Peprotech	100-3
IGF	Peprotech	100-11
Knockout DMEM	Thermo Fisher	10829018
Knockout Serum Replacement	Thermo Fisher	10828028
Laminin	Sigma Aldrich	L2020
mTeSR1	Stemcell Technologies	85850	hESC culture medium
N2 supplement	Thermo Fisher	17502001
NEAA	Thermo Fisher	11140050
Neurobasal	Thermo Fisher	21103049
Poly-L-ornithine	Sigma Aldrich	P3655
ROCK inhibitor	Tocris	1254
SB431542	Tocris	1614
SHH	Applied Biological Materials	Z200617
Stemdiff Neural Progenitor medium	Stemcell Technologies	5833	NPC expansion medium