Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Summary

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Abstract

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

Introduction

In vitro neuronal cultures have been used as a fundamental tool for studies of neurological diseases. Primary animal (mostly rodent) neural cultures^1,2 and human neural cell lines derived from gliomas or other tumors are the most commonly used in such studies. However, it has been recognized that there are significant differences between rodent and human cells. Many findings based on rodents cannot be translated to humans. Furthermore, with the rapid developments in analyzing mass genomic information and the relatively easy gene editing and whole genome sequencing, the trend is more and more geared to discovering disease prone genes and ....

Protocol

1. Enrichment of Hematopoietic Progenitor Cells from Adult Whole Blood

NOTE: Hematopoietic progenitor cells or CD34+ cells can be purified from peripheral blood mononuclear cells (PBMCs) derived from a variety of sources including cord blood, leukapheresis material and whole blood using density gradient centrifugation based methods. The method listed here uses whole blood as an example.

1. Isolation of PBMCs from Whole Blood:
   1. Add 4.5 ml of lymphocyte separation medium to the SepMate tube by pipetting it through the central hole of the insert.
   2. Dilute the blood sample with an equal volume of sterile DPBS contai....

Results

The quality of CD34 cells is critical for the success of neural stem cell transduction. High quality CD34 cells proliferate during the first couple of days of culture and appear as non adherent, homogenously round cells, floating just above the bottom of culture vessels (Figure 1A). The successful infection results in cell aggregates, which expands over time (Figure 1B), while non specific cell aggregates may occur during cell culture, that expand and the associations are loose. Adherent.......

Discussion

A detailed protocol to directly generate neural stem cells from peripheral hematopoietic progenitor cells is provided.

Compared to fibroblast cells, human peripheral blood is more accessible. Using the presented protocol, more than 1 x 10⁵ hematopoietic progenitor cells, or CD34 positive cells, can be enriched from 10 ml of whole blood. Although contamination with platelets is usually not preventable, it can be easily reduced by lower speed centrifugation and it does not interfere w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lymphocyte separation medium	Lonza	17-829E	1x
SepMate	Stemcell Technologies	15415	15 ml
Human serum	Invitrogen	34005-100
Antibiotics	Gibco	15240-062	1%
CD34 MultiSort Kit	Miltenyi Biotec	130-056-701
EDTA	Cellgro	46-034-Cl	2 mM
MACS LS column	Miltenyi Biotec	130-042-401
StemSpan SFEM medium	Stemcell Technologies	9650	1x
IMDM	Quality Biologicals	112-035-101	1x
TPO	Peprotech	300-18	100 ng/ml
Flt-3	Peprotech	300-19	100 ng/ml
SCF	Peprotech	300-07	100 ng/ml
IL-6	Peprotech	200-06	20 ng/ml
IL-7	Peprotech	200-07	20 ng/ml
IPS Sendai Reprogramming Kit	Life Technologies	A1378001
Cell scraper	Sarstedt	83.183
DMSO	Sigma	D2650
CryoTube vials	Thermo	368632
Mr. Frosty container	Thermo	5100-0001
DMEM/F12	Life Technologies	12400-024	1x
N2 supplement	Life Technologies	17502-048	1x
Bovine serum albumin	Sigma	A2934	0.1% (w/v)
bFGF	Peprotech	100-18B	20 ng/ml
EGF	Peprotech	AF-100-15
B27 supplement	Life Technologies	17504-044	1x
NSC serum free medium	Life Technologies	A1050901	1x
Poly-D-lysine/laminin coated cover slips	BD Bioscences	354087
cAMP	Sigma	A9501	300 ng/ml
Vitamin C	Sigma	A0278	0.2 mM
BDNF	Peprotech	450-02	10 ng/ml
GDNF	Peprotech	450-10	10 ng/ml
Poly-L-ornithine	Sigma	P4957	1x
PDGF-AA	Peprotech	100-13A	10 ng/ml
NT-3	Peprotech	450-03	2 ng/ml
Shh	Peprotech	1314-SH/CF	2 ng/ml
T3	Sigma	T6397	3 nM
PFA	Sigma	P6148	4%
PBS	Quality Biological	119-069-101	1x
Goat serum	Sigma	G9023	4%
TritonX-100	Sigma	T9284
Mouse monoclonal anti-Nestin	Millipore	AB5922	1:1,000 dilution
Anti-SOX2 antibody	Applied Stemcell	ASA0120	Ready to use
Mouse anti-βIII-tubulin antibody	Promega	G712A	1:1,000 dilution
Rabbit anti-GFAP antibody	Sigma	G4546	1:100 dilution
Anti-O4 antibody	R&D Systems	MAB1326	IgM; 1 ng/ml
Alexa Fluor 594 goat anti-rabbit antibody	Life techniologies	A11012	1:400 dilution
Alexa Fluor 488 goat anti-mouse antibody	Life techniologies	A11001	1:400 dilution
Alexa Fluor 488 goat anti-mouse IgM antibody	Life techniologies	A21042	1:250 dilution
DAPI	Sigma	D9542	1 μg/ml