Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Summary

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson's disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Abstract

Parkinson's disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

Introduction

Parkinson's disease (PD) is a common neurodegenerative disorder, caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM), with a prevalence of more than 1% in population over 60 years of age^1,2. Over the past decade, cell therapy, aimed at either replacing the degenerative or damaged cells, or nourishing the microenvironment around the degenerating neurons, has shown potential in treatment of PD³. Meanwhile, reprogramming technology has made significant progress⁴, which provides a prom....

Protocol

All procedures must follow the guidelines of institutional human research ethics committee. Informed consent must be obtained from patients or healthy volunteers before blood collection. This protocol was approved by the institution's human research ethics committee and was performed according to the institution's guidelines for care and use of animals.

1. Collection, isolation and expansion of PBMNCs

1. Collection of PBMNCs
   1. Collect 10-20 mL of donor’s peripheral venous blood by venipuncture with a sodium heparin preservative vial.
      NOTE: Blood samples should be stored or shipped at....

Results

Here, we report a protocol that covers different stages of iNSC-DA cell therapy to treat PD models. Firstly, PBMNCs were isolated and expanded, and reprogrammed into iNSCs by SeV infection. A schematic representation of the procedures with PBMNC expansion and iNSC induction is shown in Figure 1. On day -14, PBMNCs were isolated by using a density gradient medium (Table of Materials). Before centrifugation, blood diluted with PBS and the density gradient medium were separated.......

Discussion

Here we presented a protocol that covered different stages of iNSC-DA cell therapy for PD models. Critical aspects of this protocol include: (1) isolation and expansion of PBMNCs and reprogramming of PBMNCs into iNSCs by SeV infection, (2) differentiation of iNSCs to DA neurons, (3) establishment of unilateral 6-OHDA-lesioned PD mouse models and behavioral assessment, and (4) cell transplantation of DA precursors and behavioral assessment.

In this protocol, the first part involves collecting a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15-ml conical tube	Corning	430052
1-Thioglycerol	Sigma-Aldrich	M6145	Toxic for inhalation and skin contact
24-well plate	Corning	3337
50-ml conical tube	Corning	430828
6-OHDA	Sigma-Aldrich	H4381
6-well plate	Corning	3516
Accutase	Invitrogen	A11105-01	Cell dissociation reagent
Apomorphine	Sigma-Aldrich	A4393
Ascorbic acid	Sigma-Aldrich	A92902	Toxic with skin contact
B27 supplement	Invitrogen	17504044
BDNF	Peprotech	450-02	Brain derived neurotrophic factor
Blood collection tubes containing sodium heparin	BD	367871
BSA	yisheng	36106es60	Fetal bovine serum
cAMP	Sigma-Aldrich	D0627	Dibutyryladenosine cyclic monophosphate
CellBanker 2	ZENOAQ	100ml	Used as freezing medium for PBMNCs
Chemically defined lipid concentrate	Invitrogen	11905031
CHIR99021	Gene Operation	04-0004
Coverslip	Fisher	25*25-2
DAPI	Sigma-Aldrich	D8417-10mg
DAPT	Sigma-Aldrich	D5942
Dexamethasone	Sigma-Aldrich	D2915-100MG
DMEM-F12	Gibco	11330
DMEM-F12	Gibco	11320
Donkey serum	Jackson	017-000-121
EPO	Peprotech	100-64-50UG	Human Erythropoietin
FGF8b	Peprotech	100-25
Ficoll-Paque Premium	GE Healthcare	17-5442-02	P=1.077, density gradient medium
GDNF	Peprotech	450-10	Glial derived neurotrophic factor
GlutaMAX	Invitrogen	21051024	100 × Glutamine stock solution
Ham's-F12	Gibco	11765-054
HBSS	Invitrogen	14175079	Balanced salt solution
Human leukemia inhibitory factor	Millpore	LIF1010
Human recombinant SCF	Peprotech	300-07-100UG
IGF-1	Peprotech	100-11-100UG	Human insulin-like growth factor
IL-3	Peprotech	200-03-10UG	Human interleukin 3
IMDM	Gibco	215056-023	Iscove's modified Dulbecco's medium
Insulin	Roche	12585014
ITS-X	Invitrogen	51500-056	Insulin-transferrin-selenium-X supplement
Knockout serum replacement	Gibco	10828028	Serum free basal medium
Laminin	Roche	11243217001
Microsyringe	Hamilton	7653-01
N2 supplement	Invitrogen	17502048
NEAA	Invitrogen	11140050	Non-essential amino acid
Neurobasal	Gibco	10888	Basic medium
PDL	Sigma-Aldrich	P7280	Poly-D-lysine
SAG1	Enzo	ALX-270-426-M01
SB431542	Gene Operation	04-0010-10mg	Store from light at -20?
Sendai virus	Life Technologies	MAN0009378
Sucrose	baiaoshengke
TGFβ?	Peprotech	100-36E	Transforming growth factor  β?
Transferrin	R&D Systems	2914-HT-100G
Triton X 100	baiaoshengke		Nonionic surfactant
Trypan blue	Gibco	T10282
Xylazine	Sigma-Aldrich	X1126