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

摘要

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.

摘要

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.

引言

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....

方案

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....

结果

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.......

讨论

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.......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
15 ml 锥形管	Corning	430052
1-硫代甘油	Sigma-Aldrich	M6145	对吸入和皮肤接触有毒
24 孔板	Corning	3337
50 ml 锥形管	康宁	430828
6-OHDA	Sigma-Aldrich	H4381
6 孔板	康宁	3516
Accutase	Invitrogen	A11105-01	细胞解离试剂
阿扑吗啡	Sigma-Aldrich	A4393
抗坏血酸	Sigma-Aldrich	A92902	皮肤接触有毒
B27 补充剂	Invitrogen	17504044
BDNF	Peprotech	450-02	脑源性神经营养因子
含有肝素钠	BD	367871
BSA	益生	36106es60	胎牛血清
cAMP	Sigma-Aldrich	D0627	二丁酰腺苷环磷酸
CellBanker 2	ZENOAQ	100ml	用作 PBMNCs 的冷冻培养基
化学定义的脂质浓缩	Invitrogen	11905031
CHIR99021	基因作	04-0004
盖玻片	Fisher	25*25-2
DAPI	Sigma-Aldrich	D8417-10mg
DAPT	Sigma-Aldrich	D5942
地塞米松	Sigma-Aldrich	D2915-100MG
DMEM-F12	Gibco	11330
DMEM-F12	Gibco	11320
驴血清	Jackson	017-000-121
EPO	Peprotech	100-64-50UG	人促红细胞生成素
FGF8b	Peprotech	100-25
Ficoll-Paque Premium	GE Healthcare	17-5442-02	P=1.077，密度梯度培养基
GDNF	Peprotech	450-10	神经胶质衍生神经营养因子
GlutaMAX	Invitrogen	21051024	100 &谷氨酰胺储备液
Ham's-F12	Gibco	11765-054
HBSS	Invitrogen	14175079	平衡盐溶液
人白血病抑制因子	Millpore	LIF1010
人重组 SCF	Peprotech	300-07-100UG
IGF-1	Peprotech	100-11-100UG	人胰岛素样生长因素
IL-3	Peprotech	200-03-10UG	人白细胞介素 3
IMDM	Gibco	215056-023	Iscove 改良的 Dulbecco 培养基
罗	氏胰岛素
ITS-X	Invitrogen	51500-056	胰岛素-转铁蛋白-硒-X 添加剂
基因敲除血清替代物	Gibco	10828028	无血清基础培养基
层粘连蛋白	Roche
Microsyringe	Hamilton	7653-01
N2 补充剂	Invitrogen	17502048
NEAA	Invitrogen	11140050	非必需氨基酸
Neurobasal	Gibco	10888	碱性培养基
PDL	Sigma-Aldrich	P7280	多聚-D-赖氨酸
SAG1	Enzo	ALX-270-426-M01
SB431542	基因作	04-0010-10mg	避光储存在 -20 度？
仙台病毒	Life Technologies	MAN0009378
蔗糖	baiaoshengke
TGFβ？	Peprotech	100-36E	转化生长因子  β？
转铁蛋白	R&D Systems	2914-HT-100G
Triton X 100	百奥盛科		非离子表面活性剂
台盼蓝	Gibco	T10282
甲苯噻嗪	Sigma-Aldrich	X1126