Generation of Neuronal Cells from Blood-Derived Pluripotent Stem Cells

Abstract

Source: Becker-Kojić, Z. A., et al. GM-Free Generation of Blood-Derived Neuronal Cells. J. Vis. Exp. (2021).

This video demonstrates a technique to generate neuronal cells from blood-derived pluripotent stem cells (BD-PSCs). The BD-PSCs are obtained via reprogramming blood progenitor cells. The undifferentiated BD-PSCs are cultured on cell culture substrate-coated coverslips using a neural induction medium and a neural differentiation medium, triggering differentiation into cells with a neuronal phenotype.

Protocol

Ethic approvals were obtained when performing the experiments.

1. Isolation of human peripheral blood mononuclear cells (PBMNCs)

1. Ensure that all donors signed informed consent before blood withdrawing in compliance with institutional guidelines.
2. Take 30 mL of blood from healthy donors by trained medical personnel according to the standard protocol.
3. Isolate PBMNCs by density gradient media. Use 10 mL of media with 25 mL of 1:1 blood diluted with phosphate-buffered saline (PBS), and centrifuge at 300 x g for 30 min.
4. Isolate the interphase layer between the plasma an....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin Fraction V	Roth	T8444.4
AO/PI Cell Viability kit	Biozym	872045	Biozym discontinued. The product produced by Logos Biosystems.
Ascorbic acid 2-phosphate sequimagnes	Sigma Aldrich	A8960-5G
B27 Serum free 50x	Fisher Scientific (Gibco)	11530536
Basic FGF solution	Fisher Scientific (Gibco)	10647225
Biocoll	Merck Millipore	L6115-BC	density gradient media
BSA Frac V 7.5%	Gibco	15260037
CD45 MicroBeads	Miltenyi	130-045-801	nano-sized magnetic beads
Cell counting slides Luna	Biozym	872010	Biozym discontinued. The product produced by Logos Biosystems.
D-MEM/F12	Merck Millipore	FG4815-BC
FBS	Merck Millipore	S0115/1030B	Discontinued. Available under: TMS-013-B
GDNF recombinant human	Fisher Scientific (Gibco)	10679963
GlutaMax 100x	Gibco	35050038	L-glutamine
Heparin sodium cell	Sigma-Aldrich	H3149-50KU
Human BDNF	Fisher Scientific (Gibco)	11588836
Iscove (IMDM)	Biochrom	FG0465
Laminin mouse	Fisher Scientific (Gibco)	10267092
Luna FL Automated Cell Counter	Biozym	872040	Biozym discontinued. The product produced by Logos Biosystems.
MACS Buffer	Miltenyi	130-091-221
MEM NEAA 100x	Gibco	11140035
MiniMACS Trennsäulen	Miltenyi	130-042-201
Multiplatte Nunclon 4 wells	Fisher Scientific	10507591
N2 Supplement 100x	Fisher Scientific (Gibco)	11520536
Neurobasal Medium	Gibco	10888022
PBS sterile	Roth	9143.2
Poly-L-ornithine	Sigma-Aldrich	P4957-50ML