GM-Free Generation of Blood-Derived Neuronal Cells

Summary

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Abstract

Many human neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

Introduction

Development of basic and pre-clinical stem cell research methods encourages the clinical application of stem cell-based therapies for neurological diseases. Such potential therapy critically depends on the method for generation of human neural cells leading to functional recovery¹.

Neural stem cells (NSCs) self-renew and differentiate into new neurons throughout life in a process called adult neurogenesis. Only very restricted brain areas harbor NSCs competent to generate newborn neurons in adulthood. Such NSCs can give rise to mature neurons, which are involved in learning and memory, thus replacing lost or damaged ....

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 buffer saline (PBS), and centrifuge at 300 x g for 30 min.
4. Isolate the interphase layer between the plasma and the density gradient media ....

Results

The results provide evidence that this novel GM-free method is capable of reverting blood progenitor cells to their most primitive state without directly acting on the human genome.

We have previously shown that GPI-linked protein specific antibody crosslinking initiates via PLCγ/IP3K/Akt/mTOR/PTEN upregulation of highly conserved developmentally relevant genes such as WNT, NOTCH and C-Kit, thus initiating a process of dedifferentiation that leads to the first step to generation of HS.......

Discussion

The non-GM method of reprogramming human cells described in this work is based on membrane to nucleus activation of signaling(s) machinery behind the GPI-linked human membrane glycoprotein that initiates the process of dedifferentiation leading to the ex vivo generation and expansion of self-renewing PSCs obtained from non-manipulated human peripheral blood. These cells when cultured in appropriate media are capable of re-differentiation into cells belonging to different germ layers⁶.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Albumin Fraction V	Roth	T8444.4
Anti-GFAP Cy3 conjugate	Merck Millipore	MAB3402C3
Anti-MAP2 Alexa Fluor 555	Merck Millipore	MAB3418A5
Anti-Nestin Alexa Fluor 488	Merck Millipore	MAB5326A4
Anti-Tuj1 Alexa Fluor 488	BD Pharmingen	560381
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.
Chamber Slides Lab-Tek	Fisher Scientific	10234121
D-MEM/F12	Merck Millipore	FG4815-BC
Durcupan	Sigma Aldrich	44610	epoxy resin
FBS	Merck Millipore	S0115/1030B	Discontinued. Available under: TMS-013-B
GDNF recombinant human	Fisher Scientific (Gibco)	10679963
GlutaMax 100x	Gibco	35050038	L-glutamine
Glutaraldehyde grade	Sigma-Aldrich	G5882-50ML
Heparin sodium cell	Sigma-Aldrich	H3149-50KU
Human BDNF	Fisher Scientific (Gibco)	11588836
Iscove (IMDM)	Biochrom	FG0465
Laminin mouse	Fisher Scientific (Gibco)	10267092
Lead citrate	Sigma-Aldrich	15326-25G
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
Morada digital camera	Olympus
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
Super Glue-3 Loctite	Henkel
TEM FEI Technai G2 Spirit	FEI Europe
Ultracut UC-6	Leica
Uranyl acetate C	EMS	22400