Name: generation of induced neural stem cells from peripheral mononuclear cells and differentiation toward dopaminergic neuron precursors for transplantation studies
Uploaded: 2019-07-11T14:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studie

The work was supported by the following grants: Stem Cell and Translation National Key Project (2016YFA0101403), National Natural Science Foundation of China (81661130160, 81422014, 81561138004), Beijing Municipal Natural Science Foundation (5142005), Beijing Talents Foundation (2017000021223TD03), Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five&#8211;year Plan (CIT &#38; TCD20180333), Beijing Medical System High Level Talent Award (2015-3-063), Beijing Municipal Health Commission Fund (PXM 2018_026283_000002), Beijing One Hundred, Thousand, and Ten Thousand Talents Fund (2018A03), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201706), and the Royal Society-Newton Advanced Fellowship (NA150482).

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&#39;s human research ethics committee and was performed according to the institution&#39;s guidelines for care and use of animals.
1. Collection, isolation and expansion of PBMNCs
<ol>
	<li>Collection of PBMNCs
	<ol>
		<li>Collect 10-20 mL of don...

<ol>
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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...

Parkinson&#39;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 age1,2. 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 promising cellular source for replacement therapy. Human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) have been proven to be able to differentiate into DA neural cells, which could survive, maturate, and improve the motor functions when grafted into rat and non-human primate PD models5,6,7,8. iPSCs represent a milestone in cellular reprogramming technologies and have a great potential in cell transplantation; however, there is still a concern about the risk of tumor formation from the incompletely differentiated cells. An alternative cellular source for cell transplantation is lineage-committed adult stem cells obtained through direct reprogramming, such as induced neural stem cells (iNSCs), which can be derived from the unstable intermediates, bypassing the pluripotency stage9,10,11.
Both iPSCs and iNSCs can be reprogrammed from autologous cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells12,13,14, thus reducing the immunogenicity of transplanted cells to a great degree. Moreover, compared with iPSCs, iNSCs are inherent with reduced risk of tumor formation and lineage-committed plasticity, only able to differentiate into neurons and glia11. In the initial studies, human or mouse iPSCs and iNSCs were generated from fibroblasts obtained from skin biopsies, which is an invasive procedure14,15. With this respect, PBMNCs are an appealing starter cell source because of the less invasive sampling process, and the ease to obtain large numbers of cells within a short period of expansion time16. Initial reprogramming studies employed integrative delivery systems, such as lentiviral or retroviral vectors, which are efficient and easy to implement in many types of cells17; however, these delivery systems may cause mutations and reactivation of residual transgenes, which present safety issues for clinical therapeutic purposes12. Sendai virus (SeV) is a non-integrative RNA virus with a negative-sense, single-stranded genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming18,19. Recombinant SeV vectors are available that contain reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC in their open reading frames. In addition, SeV viral vectors can be further improved by introducing temperature-sensitive mutations, so that they could be rapidly removed when the culture temperature is raised to 39 &#176;C20. In this article, we describe a protocol to reprogram PBMNCs to iNSCs using the SeV system.
Many studies have reported derivation of DA neurons from human ESCs or iPSCs using various methods6,8,21. However, there is a shortage of protocols describing the differentiation of DA neurons from iNSCs in details. In this protocol, we will describe the efficient generation of DA neurons from iNSCs using a two-stage method. The DA neuronal precursors can be transplanted into the striatum of PD mouse models for safety and efficacy evaluations. This article will present a detailed protocol that covers various stages from generation of induced neural stem cells by Sendai virus, differentiation of iNSCs into DA neurons, establishment of mouse PD models, to transplantation of DA precursors into the striatum of the PD models. Using this protocol, one can generate iNSCs from patients and healthy donors and derive DA neurons that are safe, standardizable, scalable and homogeneous for cell transplantation purposes, or for modeling PD in a dish and investigation of the mechanisms underlying disease onset and development.

<table border="0" cellpadding="0" cellspacing="0" style="width:828px;" width="827">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">15-ml conical tube</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">430052</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1-Thioglycerol</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">M6145</td>
			<td>Toxic for inhalation and skin contact</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">24-well plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">3337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">50-ml conical tube&#160;</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">6-OHDA</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">H4381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">6-well plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:153px;">3516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Accutase</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">A11105-01</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Apomorphine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">A4393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ascorbic acid</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">A92902</td>
			<td>Toxic with skin contact&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">B27 supplement&#160;</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">BDNF</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">450-02</td>
			<td>Brain derived neurotrophic factor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">Blood collection tubes containing sodium heparin</td>
			<td style="width:183px;">BD</td>
			<td style="width:153px;">367871</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">BSA</td>
			<td style="width:183px;">yisheng</td>
			<td style="width:153px;">36106es60</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">cAMP</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D0627</td>
			<td>Dibutyryladenosine cyclic monophosphate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">CellBanker 2</td>
			<td style="width:183px;">ZENOAQ</td>
			<td style="width:153px;">100ml</td>
			<td>Used as freezing medium for PBMNCs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Chemically defined lipid concentrate</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">11905031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">CHIR99021</td>
			<td style="width:183px;">Gene Operation</td>
			<td style="width:153px;">04-0004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Coverslip</td>
			<td style="width:183px;">Fisher</td>
			<td style="width:153px;">25*25-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPI</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D8417-10mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DAPT</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D5942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Dexamethasone</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">D2915-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DMEM-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">DMEM-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Donkey serum</td>
			<td style="width:183px;">Jackson</td>
			<td style="width:153px;">017-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">EPO</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-64-50UG</td>
			<td>Human Erythropoietin</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">FGF8b</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-25</td>
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			<td height="21" style="height:21px;width:277px;">Ficoll-Paque Premium</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:153px;">17-5442-02</td>
			<td>P=1.077, density gradient medium</td>
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			<td height="21" style="height:21px;width:277px;">GDNF</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">450-10</td>
			<td>Glial derived neurotrophic factor</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">GlutaMAX</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">21051024</td>
			<td>100 &#215; Glutamine stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Ham&#39;s-F12</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">11765-054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">HBSS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">14175079</td>
			<td>Balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Human leukemia inhibitory factor</td>
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			<td style="width:153px;">300-07-100UG</td>
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			<td height="21" style="height:21px;width:277px;">IGF-1</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-11-100UG</td>
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			<td height="21" style="height:21px;width:277px;">IL-3</td>
			<td style="width:183px;">Peprotech</td>
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			<td>Human interleukin 3</td>
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			<td style="width:153px;">215056-023</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ITS-X</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">51500-056</td>
			<td>Insulin-transferrin-selenium-X supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Knockout serum replacement</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">10828028</td>
			<td>Serum free basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Laminin</td>
			<td style="width:183px;">Roche&#160;</td>
			<td style="width:153px;">11243217001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Microsyringe</td>
			<td style="width:183px;">Hamilton</td>
			<td style="width:153px;">7653-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">N2 supplement&#160;</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">NEAA</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:153px;">11140050</td>
			<td>Non-essential amino acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Neurobasal</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">10888</td>
			<td>Basic medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">PDL</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">P7280</td>
			<td>Poly-D-lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">SAG1</td>
			<td style="width:183px;">Enzo</td>
			<td style="width:153px;">ALX-270-426-M01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">SB431542</td>
			<td style="width:183px;">Gene Operation</td>
			<td style="width:153px;">04-0010-10mg</td>
			<td>Store from light at -20&#8451;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sendai virus</td>
			<td style="width:183px;">Life Technologies</td>
			<td style="width:153px;">MAN0009378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Sucrose</td>
			<td style="width:183px;">baiaoshengke</td>
			<td style="width:153px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">TGF&#946;&#8546;</td>
			<td style="width:183px;">Peprotech</td>
			<td style="width:153px;">100-36E</td>
			<td>Transforming growth factor&#160; &#946;&#8546;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Transferrin</td>
			<td style="width:183px;">R&#38;D Systems</td>
			<td style="width:153px;">2914-HT-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Triton X 100</td>
			<td style="width:183px;">baiaoshengke</td>
			<td style="width:153px;"></td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Trypan blue</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:153px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Xylazine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:153px;">X1126</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of induced neural stem cells from peripheral mononuclear cells and differentiation toward dopaminergic neuron precursors for transplantation studies

Parkinson&#39;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.

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...

Watch this Scientific Journal Video about Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studie

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

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&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Research

JoVE Journal

Developmental Biology

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