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

Wei Zheng; Zhiguo Chen

doi:10.3791/59690

JoVE Journal > Developmental Biology

发育生物学

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

Published: July 11, 2019

doi:

10.3791/59690

Wei Zheng^1,2, Zhiguo Chen^1,2

¹Cell Therapy Center, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, and Key Laboratory of Neurodegeneration,Ministry of Education, ²Center of Neural Injury and Repair,Beijing Institute for Brain Disorders

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 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 °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.

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

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 room temperature (RT). Process the blood samples within 24 h.
Preparation of culture medium
1. Prepare serum free medium (SFM) by combining the following components: 245 mL of Iscove's modified Dulbecco's medium (IMDM), 240 mL of Ham’s F-12, 5 mL of insulin-transferrin-selenium-X supplement (ITS-X), 5 mL of 100x glutamine stock solution (Table of Materials), 5 mL of chemically defined lipid concentrate, 2.5 g of fetal bovine serum, 0.025 g of ascorbic acid and 9 μL of 1-thioglycerol. Filter the medium and store it at 4 °C.
  CAUTION: Ascorbic acid and 1-thioglycerol are toxic by skin contact and inhalation.
2. To prepare mononuclear cell (MNC) medium, supplement the SFM medium with 10 ng/mL human interleukin 3 (IL-3), 2 U/mL erythropoietin (EPO), 100 ng/mL human stem cell factor (SCF), 40 ng/mL human insulin-like growth factor 1 (IGF-1), 100 μg/mL holo-transferrin and 1 μM dexamethasone. Filter the medium and store it at 4 °C.
  NOTE: Prepare the medium immediately before use.
Isolation of PBMNCs
1. Ultraviolet-sterilize a clean bench prior to use. Sterilize all surfaces and equipment with 75% alcohol. Sterilize all tips by using an autoclave.
2. Transfer the peripheral blood (PB) into a 50 mL conical tube and dilute the PB with an equal volume of sterile Dulbecco's phosphate-buffered saline (D-PBS).
3. Prepare 15 mL of sterilized density gradient medium (Tables of Materials) in another 50 mL conical tube.
  NOTE: Keep the density gradient medium and PB at RT to allow for better isolation of PBMNCs.
4. Tilt the conical tube containing the density gradient medium at a 45° angle, and then slowly and carefully lay 30 mL of diluted PB onto the density gradient medium.
  NOTE: Take care and allow the PB to slowly run down the side of the conical tube onto the density gradient medium layer. Red blood cells will deposit to the bottom of the tube. Tilt the tube carefully to minimize disruption of the layer interface.
5. Centrifuge the tubes at 800 x g for 15 min at RT with the centrifuge brake set at "off" position. Aspirate the yellow, upper plasma layer and discard it. Then transfer the white cloudy thin film layer containing MNCs with a 10 mL pipette to a new 50 mL conical tube.
  NOTE: Switching centrifuge brake off is important for the isolation of MNCs.
6. Add 30 mL of D-PBS to the tube with MNCs and centrifuge at 600 x g for 10 min at 4 °C. Discard the supernatant, and then add 45 mL of D-PBS to re-suspend the cells. Centrifuge at 400 x g for 10 min at 4 °C.
  NOTE: The centrifuge brake should be switched on for this and the following centrifugation steps. As the cell pellets are dense, add 1-2 mL of D-PBS to gently re-suspend the pellets, and then add D-PBS to 45 mL.
7. Discard the supernatant and re-suspend the cells with 5 mL of D-PBS and count the live cells with the trypan blue exclusion method.
8. After setting aside the MNCs needed for expansion, freeze the remaining cells for future use.
  NOTE: At least 5 x 10⁶ MNCs can be frozen in one vial with 1 mL of freezing medium (Table of Materials). The protocol can be paused here.
Expansion of MNCs
1. On day -14, seed MNCs at a density of 2-3 x 10⁶ cells per milliliter in one well of six-well plates with 1.5 mL of pre-warmed (37 °C) MNC medium. Incubate at 37 °C, 5% CO₂ for 2 days.
2. On day -11, collect the cells and medium with a sterilized pipette and transfer to a new 15 mL conical tube. Centrifuge the cells at 250 x g for 5 min at RT. Discard the supernatant and re-suspend the cells in 1 mL of pre-warmed (37 °C) MNC medium.
3. Count the viable cells with trypan blue. Seed the MNCs at a density of 1 x 10⁶cells per milliliter in pre-warmed MNC medium and incubate at 37 °C, 5% CO₂for 3 days.
  NOTE: It is expected that the total number of cells may decrease on day -11.
4. On day -8, repeat steps 1.4.2-1.4.3 and culture the cells for 3 days.
5. On day -4, repeat steps 1.4.2-1.4.3 and culture the cells for 3 days.
  NOTE: After 14 days of culture, an equal or greater number of MNCs should remain in the culture.

2. Reprogramming of PBMNCs to iNSCs by SeV Infection

Preparation of solution and culture medium
1. Prepare a poly-D-lysine (PDL) stock solution by dissolving 100 mg of PDL with 100 mL of H₂O to a concentration of 1 mg/mL. Store at -20 °C in 1 mL aliquots.
2. Prepare an insulin stock solution by dissolving 100 mg of insulin in 20 mL of 0.01 N HCl to a concentration of 5 mg/mL. Store at -20 °C in 1 mL aliquots.
3. To prepare 200 mL of iNSC basal medium, combine 96 mL of DMEM-F12 and 96 mL of basic medium (Table of Materials) with 2 mL of 100x glutamine stock solution, 2 mL of nonessential amino acid (NEAA), 2 mL of N2 supplement and 2 mL of B27 supplement. Add 10 ng/mL recombinant human leukemia inhibitory factor, 3 μM CHIR99021 and 2 μM SB431542 prior to use. Filter the medium and store it at 4 °C.
  NOTE: Use the medium within 2 weeks. Add recombinant human leukemia inhibitory factor, CHIR99021 and SB431542 immediately before use.
Reprogramming of PBMNCs to iNSCs by SeV Infection
1. Ultraviolet-sterilize a clean bench prior to use. Sterilize all surfaces and equipment with 75% alcohol. Sterilize all tips using an autoclave.
2. On day 0, collect the cells in MNC medium and transfer to a 15 mL conical tube. Centrifuge the cells at 200 x g for 5 min. Aspirate the supernatant and re-suspend the cells with 1 mL of pre-warmed MNC medium.
3. Count the viable cells with trypan blue. Re-suspend the cells with pre-warmed (37 °C) MNC medium to a concentration of 2 x 10⁵ cells per well in 24-well plates.
4. After removing the SeV tubes from -80 °C storage, thaw the tubes containing SeV in 37 °C water bath for 5-10 s, and then allow them to thaw at RT. Once thawed, place them on ice immediately.
5. Add the SeV encoding human Klf4, Oct3/4, SOX2 and c-MyC to the wells, at a multiplicity of infection (MOI) of 10. Centrifuge cells with plates at 1,000 x g for 30 min to facilitate the attachment of cells. Leave the cells and supernatant in the plates. Place the plates in the incubator at 37 °C, 5% CO₂.
  CAUTION: All procedures involving SeV must be performed in a safety cabinet, and all tips and tubes should be treated with ethanol or bleach before disposal.
6. On day 1, transfer the medium and cells to a 15 mL centrifuge tube. Rinse the well with 1 mL of MNC medium. Centrifuge the cell suspension at 200 x g for 5 min. Aspirate the supernatant and re-suspend the cells with 500 μL of fresh pre-warmed MNC medium in 24-well plates.
  NOTE: Use a low attachment 24-well plate to prevent attachment of any cells before plating on PDL/laminin.
7. On day 2, dilute 1 mL of 1 mg/mL PDL with 19 mL of D-PBS to a concentration of 50 μg/mL. Coat 6-well plates with 50 μg/mL PDL for at least 2 h at RT.
8. Dilute 200 μL of 0.5 mg/mL laminin with 20 mL of D-PBS to a concentration of 5 µg/mL. Aspirate PDL in the 6-well plates, and dry on the vertical clean bench.
9. Coat 6-well plates with 5 μg/mL laminin and incubate for 4-6 h at 37 °C. Wash with D-PBS before use.
10. On day 3, plate the transduced cells obtained in step 2.2.6 in iNSC medium on PDL/laminin-coated 6-well plates.
  NOTE: Move the plates gently if needed after the cells are placed on PDL/laminin-coated plates, trying not to disturb the attachment of the cells.
11. On day 5, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently.
  NOTE: It is expected that the cells will undergo drastic death (>60%).
12. On day 7, add 1 mL of pre-warmed (37 °C) iNSC medium in each well in 6-well plates gently.
13. From day 9 to day 28, replace spent medium with fresh pre-warmed (37 °C) iNSC medium every day. Monitor the emergence of iNSC colonies. Pick and transfer iNSC clones for expansion in about 2-3 weeks. Pick up colonies with appropriate morphology using burned glass pipettes, excluding any possibly contaminating cells, and aspirate the colonies with 200 μL tips.
  NOTE: The characterized iNSCs can be frozen for future use with 2-5 colonies in one vial. The freezing medium includes serum free basal medium (Table of Materials) and dimethyl sulfoxide mixed at a ratio of 9:1, which should be prepared immediately before use. The protocol can be paused here.

3. Differentiation of iNSCs to dopaminergic neurons

Preparation of solution and culture medium
1. Prepare 200 mL of iNSC differentiation basal medium by combining 192 mL of DMEM-F12 with 2 mL of 100x glutamine stock solution, 2 mL of NEAA, 2 mL of N2 supplement and 2 mL of B27 supplement.
  NOTE: Use the medium within 2 weeks.
2. Prepare iNSC differentiation stage I medium by supplementing the iNSC differentiation basal medium with 1 μM SAG1 and 100 ng /mL FGF8b.
  NOTE: Use the medium within 2 weeks.
3. Prepare iNSC differentiation stage II medium by supplementing the iNSC differentiation basal medium with 0.5 mM cyclic adenosine monophosphate (cAMP), 0.2 mM ascorbic acid, 10 μM DAPT, 10 ng/mL brain derived neurotrophic factor (BDNF), 10 ng/mL glial derived neutrophic factor (GDNF) and 1 ng/mL transforming growth factor βIII (TGF-βIII).
  NOTE: Use the medium within 2 weeks.
Coating the culture dishes
1. Ultraviolet-sterilize a clean bench prior to use. Sterilize all surfaces and equipment with 75% alcohol. Sterilize all tips using an autoclave.
2. For PDL coating, at least one day before re-plating the cells, dilute 1 mL of 1 mg/mL PDL with 19 mL of D-PBS to a concentration of 50 μg/mL. Coat 12 mm glass coverslips that have been sterilized with 75% alcohol in the 24-well plates with 50 μg/mL PDL at RT for at least 2 h.
3. For laminin coating, dilute 200 μL of 0.5 mg/mL laminin with 20 mL of D-PBS to a concentration of 5 μg/mL. Aspirate PDL and dry the wells in the clean bench. Coat the 12 mm coverslips with 5 μg/mL laminin and incubate for 4-6 h at 37 °C. Wash with D-PBS before use.
Passage cells for differentiation.
1. When the confluence of cultured iNSCs reaches 70-90%, aspirate medium from the culture plate, and add 1 mL of D-PBS to wash the cells. Add 1 mL of pre-warmed (37 °C) cell dissociation reagent (Table of Materials) per well and incubate at 37 °C for 3 min to dissociate the cells.
2. After incubation for 3 min, the cells have become semi-floating; add 3 mL of pre-warmed (37 °C) DMEM-F12 medium per well, and pipette cells up and down to dissociate cell pellets into single cells.
3. Transfer cells into a 15 mL conical tube, and centrifuge at 250 x g for 3 min. Aspirate the supernatant, re-suspend the cells with appropriate volume of pre-warmed (37 °C) iNSC medium according to the number of cells.
4. Count the cells using the trypan blue exclusion method. Plate 5 x 10³ cells per 12 mm glass coverslip in 24-well plates and incubate at 37 °C, 5% CO₂.
Differentiate iNSCs into dopaminergic neurons.
1. Start differentiation 24 h after re-plating the cells onto PDL/laminin-coated coverslips. Aspirate culture medium, wash cells once with D-PBS, and then add 600 μL of pre-warmed differentiation stage I medium per well in 24-well plates and incubate at 37 °C, 5% CO₂.
2. Change medium every day from day 1 to day 10 during the first stage of differentiation.
3. On day 10, aspirate the culture medium, and wash cells once with D-PBS. Add 600 μL of pre-warmed (37 °C) differentiation stage II medium per well in 24-well plates and incubate at 37 °C, 5% CO₂.
4. Change medium every other day from day 11 to day 25 during the second stage of differentiation. The differentiated cells can be fixed by paraformaldehyde at different time points for analysis.
5. For immunofluorescent staining, wash the cells with D-PBS three times gently at chosen time points within differentiation day 11 to 25.
6. Pipette 300 μL of nonionic surfactant (Table of Materials) into 100 mL of PBS to make a 0.3% nonionic surfactant in PBS.
  CAUTION: Nonionic surfactant is toxic by skin contact and inhalation.
7. Fix the cells with 4% paraformaldehyde for 10 min at RT. Then wash with 0.3% in PBS three times.
  CAUTION: Paraformaldehyde is toxic by skin contact and inhalation.
8. Block the cells by 3% donkey serum for 2 h at RT.
9. Dilute the primary antibody in 1% donkey serum at an appropriate concentration and gently triturate to mix. Add 300 μL of the primary antibody solution to each well of the 24-well plate. Incubate the cells at 4 °C overnight. Wash the cells with 0.3% nonionic surfactant in PBS three times.
10. Dilute the secondary antibody in 1% donkey serum at an appropriate concentration and gently triturate to mix. Add 300 μL of the secondary antibody solution to each well of the 24-well plate. Incubate the cells at RT for 2 h, protected from light.
11. Wash the cells with 0.3% nonionic surfactant in PBS three times. Dilute 4',6-diamidino-2-phenylindole (DAPI) with PBS with 1:500 dilution. Incubate the cells in each well of the 24-well plate with 300 μL of diluted DAPI for 15 min at RT, protected from light. Wash the cells with 0.3% nonionic surfactant in PBS three times.
12. Gently take out the coverslips from the wells of plates with forceps. Dry in dark overnight at RT. Mount under a fluorescence microscope.

4. Establishment of unilateral 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models

To generate PD mouse models for cell transplantation, use adult male SCID-beige mice weighing 20-25 g for 6-OHDA injection.
Preparation of drugs for surgery
1. Prepare a 0.2% ascorbic acid solution by dissolving 0.2 g of ascorbic acid into 100 mL of sterilized saline (0.9%) and store at -80 °C until use. On the day of surgery, dilute 0.2% ascorbic acid solution by 10 times to obtain a 0.02% ascorbic acid solution.
  NOTE: Ascorbic acid is added to prevent oxidation of 6-OHDA to an inactive form.
2. To prepare a 6-OHDA solution, weigh appropriate amount of 6-OHDA into a sterilized 1 mL tube, and then add some volume of 0.02% ascorbic acid to make a 5 μg/μL 6-OHDA solution. Vortex the mixture until it is dissolved. Place the 6-OHDA on ice until use.
  NOTE: 6-OHDA is temperature and light sensitive. Be careful to protect the solution from light and keep it on ice before use.
Prepare sterilized surgical equipment by autoclaving before surgery. Clean all equipment and surface areas with ethanol when setting up the stereotaxic frame. Set up a mouse recovery cage under a heating lamp.
Conduct surgery to establish unilateral 6-OHDA-lesioned mouse models.
1. Weigh each mouse, record the weight and calculate the amount of drug that needs to be administered. Each mouse receives 0.5 mg/kg atropine 20 min prior to operations. Anesthetize the mouse with 80 mg/kg ketamine and 10 mg/kg xylazine.
2. Administer 0.5 mg/kg atropine by intraperitoneal injection.
3. Anesthetize the mouse with 80 mg/kg ketamine and 10 mg/kg xylazine by intraperitoneal injection 20 min after administration of atropine.
4. Put the mouse in a closed chamber. After 3-5 min, the mouse will be deeply anesthetized without response to hind leg pinch.
  NOTE: It is expected that the mouse that has received anesthesia would experience an excitation period.
5. Shave the head of mouse and apply erythromycin eye ointment on the eyes of mouse for protection from developing corneal ulcers.
6. Place the mouse on the stereotaxic apparatus. Fix the mouse with incisor bars firstly. Insert the ear cups correctly to make the mouse head in a flat and secure position.
7. Sterilize the head of the mouse with povidone iodine and isopropyl alcohol. Cut a sagittal incision (~1.5 cm) on the head skin with a scalpel blade, and expose the skull. Adjust the incisor bar and ear bars to reduce the height difference between bregma and lambda to less than 0.1 mm.
  NOTE: The mouse bregma is located at the intersection of coronal and sagittal sutures, and lambda is at the intersection of lambdoid and sagittal sutures.
8. Slowly move and lower the tip of the needle towards bregma and treat the bregma as a zero point. Move the tip to a position with coordinates of A/P +0.5 mm, M/L -2.1 mm relative to bregma. Retract the tip and mark the point. Burr a little hole into the skull.
9. Extract 2 μL of 5 μg/μL 6-OHDA solution into the microsyringe (Table of Materials). Return the needle to the point marked, and insert the needle to D/V -3.2 mm.
10. Inject 2 μL of 5 μg/μL 6-OHDA solution (10 μg total) at a rate of 1 μL/min. After injection of 6-OHDA is completed, leave the needle in place for another 5 min. Then retract the injection needle slowly.
11. Close the incision with sutures and apply erythromycin eye ointment on the eyes of mouse. Deliver 0.5 mL saline subcutaneously to prevent dehydration, and apply an antibiotic ointment directly on the sutured skin.
12. Remove the mouse from stereotaxic apparatus and put it in the recovery cage. Put the mouse back and allow access to food and water until it regains consciousness. Treat the mouse with analgesic in drinking water daily post-surgery for 2-3 days, and a recommended dosage of Ibuprofen is 0.03 mg/g of body weight per day.
13. Inspect the mouse daily post-surgery.

5. Behavioral assessment after unilateral 6-OHDA lesioning

Two to three weeks following surgery, conduct behavioral assessment to estimate PD symptoms. Weigh each mouse, record their weight and calculate the amount of drug that should be administered (0.5 mg/kg apomorphine prior to assessment).
Administer 0.5 mg/kg apomorphine by subcutaneous injection before assessment and place the mouse in a glass cylinder.
After a 5 min habituation period, count the number of contralateral and ipsilateral rotations relative to the lesion side per minute and record their activity with a video camera.
Mice with contralateral minus ipsilateral rotations >7 rpm/min are considered successfully lesioned and selected as candidates for cell transplantation experiments. Return the mice to the housing cages after a 30-min rest.
NOTE: If the mouse were successfully lesioned, 6-OHDA injected mouse will show a greater bias in turning towards contralateral side since the DA agonist activates the supersensitive denervated striatum of the lesioned side predominantly.
Conduct the behavioral assessment one week before and 2, 4, 6, 8, 12, 16 weeks after cell transplantation.

6. Cell transplantation of DA precursors

Prepare cell suspension for transplantation. For cell engraftment, suspend 2 x 10⁵ DA precursors mixed by D10 and D13 DA precursors at a ratio of 1:7 in 4 μL of 5 g L^-1 glucose in balanced salt solution (Table of Materials) of transplantation buffer.
Perform cell transplantation surgery following the procedures described in section 4.4, except that 6-OHDA is replaced by DA precursor cell suspension or buffer.
Perform the behavioral assessment 2, 4, 6, 8, 12, 16 weeks after cell transplantation of DA precursors following the procedures described in section 5. Count the number of apomorphine-induced contralateral rotations relative to the lesion side per minute and record their activity with a video camera.
NOTE: After transplantation, a reduced rate of contralateral rotations suggests an improved motor function.
At 4, 8 ,12, and 16 weeks after cell transplantation, perfuse the mouse under deep anesthesia with 4% paraformaldehyde until the body of mouse becomes stiff and the liver of mouse becomes pale.
Separate the brain of mouse gently and put the brain in 4% paraformaldehyde at 4 °C overnight.
On the second day, put the brain in 30% sucrose for dehydration until the brain sinks to the bottom.
Slice the brains at 40 μm thickness by using a freezing microtome.
Perform immunostaining as previously described in steps 3.4.5-3.4.12.

Representative 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 into two layers. After centrifugation, four gradient layers appeared (from bottom to top): the bottom layer contained granulocytes and erythrocytes; the second layer contained density gradient medium; the third contained PBMNCs (red arrow); the top layer contained platelet-rich plasma (Figure 2A). After 14 days of expansion, PBMNCs were infected with SeV as day 0. On day 1, medium with SeV was removed (Figure 2B). As illustrated in Figure 2B, it is expected that the number of cells was reduced gradually. Cells underwent a drastic death (>60%) until day 5 (Figure 2B). iNSC colonies emerged on day 12 at the earliest (Figure 2B). After picking and transferring iNSC clones for expansion for a number of passages, the morphology of cells is shown in Figure 2C. The iNSCs showed a good morphology and could self-renew stably in iNSC medium, either in a monolayer form or as spheres (Figure 2C).

iNSCs could give rise to DA neurons using a two-stage method (Figure 1). During stage one which lasted 10 days, iNSCs were treated with SAG1 and FGF8b to induce specification of VM floor plate cells with neurogenic potentials. Then the cells were treated with ascorbic acid, BDNF, GDNF, cAMP, DAPT, TGF-βIII in the second stage (Figure 1). With this two-stage method, DA precursors could be obtained towards the end of the first stage, and more mature DA neurons could be generated in the end of the second stage. After 24 days of differentiation, iNSCs could be efficiently specified to DA neurons as a majority of them expressed forkhead box A2 (FOXA2), neuron-specific class III β-tubulin (TUJ1) and tyrosine hydroxylase (TH) (Figure 3).

Three weeks after establishment of unilateral 6-OHDA-lesioned PD mouse models, behavioral assessment was conducted to estimate PD symptoms (Figure 4A). Then one week later, dopaminergic precursors were transplanted to the PD mouse models (Figure 4A). The behavioral assessment was performed one week before and 2, 4, 6, 8, 12 weeks after cell transplantation (Figure 4A). The mice that had received cell transplantation showed significant improvement in motor function (Figure 4B). The extent of 6-OHDA-induced lesioning can be verified by post-mortem immunofluorescent staining for TH at the striatum, medial forebrain bundle (MFB) and SNpc (Figure 4C). The TH-positive signals in engrafted mice were greatly recovered in the striatum where cells were implanted and mildly recovered at SNpc (Figure 4C). Three months after transplantation, about 13.84% were TH⁺ DA neurons among surviving cells (Figure 4D,E). About 91.72% and 86.76% of the TH⁺cells were expressed orphan nuclear receptor (NURR1) and FOXA2, respectively (Figure 4D,E). About 98.77% of the TH⁺ cells were co-labeled with G-protein-coupled inward rectifier potassium (GIRK2) (Figure 4D,E).

Figure 1: A schematic representation of procedures regarding PBMNC expansion, iNSC induction and differentiation of iNSCs into DA precursors. PBMNCs were isolated and expanded in MNC medium for over 14 days, and then infected with SeV encoding human SOX2, OCT3/4, c-MYC and KLF4. iNSC colonies emerged as early as 12 days after SeV infection. After 3-4 weeks, iNSC colonies were picked and differentiated into DA precursors by a two-stage method. PBMNCs: peripheral blood mononuclear cells; MNC: mononuclear cells; iNSCs: induced neural stem cells; SeV: Sendai virus; DA: dopaminergic; PDL: poly-D-lysine; BDNF: brain-derived neurotrophic factor; GDNF: glial cell line-derived neurotrophic factor; AA: ascorbic acid; cAMP: dibutyryladenosine cyclic monophosphate; TGF: transforming growth factor. Please click here to view a larger version of this figure.

Figure 2: PBMNCs are isolated and expanded, and then reprogrammed into iNSCs by SeV infection. (A) An example of PBMNCs before and after gradient centrifugation. Before centrifugation, diluted blood samples and density gradient medium were separated into two layers. After centrifugation, four density gradient layers formed from bottom to top: the bottom layer contained granulocytes and erythrocytes; the second layer contained the density gradient medium; the third layer contained PBMNCs (red arrow); the top layer contained platelet-rich plasma. (B) Images of the typical morphology of cells after PBMNCs were infected with SeV on days 1, 2, 5, and 12. After PBMNCs were infected with SeV, some of the cells died and the number of cells was reduced gradually. A small number of cells remained on day 5. On day 12, iNSCs colonies emerged. Scale bar, 100 μm. (C) The typical morphology of iNSCs of passage number 20 in monolayer and sphere culture. Scale bar, 100 μm. Please click here to view a larger version of this figure.

Figure 3: Differentiation of iNSCs to dopaminergic neurons. Immunofluorescent staining for FOXA2, TH, TUJ1, DAPI and merged images on day 24 on cells differentiated from iNSCs. Scale bar, 50 μm. Please click here to view a larger version of this figure.

Figure 4: Transplantation of iNSC-differentiated DA precursors into unilateral 6-OHDA-lesioned PD mouse models. (A) Timeline for cell transplantation and behavioral tests. (B) The results of behavioral tests at different time points from the 6-OHDA+cells group (n = 10), 6-OHDA+buffer group (n = 8) and control group (n = 3). Data are presented as mean ± standard error of the mean (SEM). ***p < 0.001 by two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. (C) Post-mortem immunofluorescent staining for TH at the striatum, medial forebrain bundle (MFB) and substantia nigra (SN) of 6-OHDA-leisioned hemisphere, 6-OHDA+cells hemisphere, and control group. Scale bars, 100 μm. (D) Percentages of FOXA2/TH, NURR1/TH, GIRK2/TH, TH/HNA in engrafted mice 3 months after transplantation. HNA: human nuclei antibody. Data are presented as mean ± SEM. (E) Immunofluorescent staining for FOXA2, NURR1, GIRK2, TH and HNA on brain slices from PD mice 12 weeks after cell transplantation. HNA: human nuclei antibody. This figure has been modified from Yuan et al.¹¹. Please click here to view a larger version of this figure.

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 and expanding PBMNCs in a serum-free medium (MNC medium), which preferentially expands erythroblasts and does not support lymphocyte proliferation. In previous studies, several somatic cell types have been reprogrammed to iPSCs or iNSCs¹²^,¹³^,¹⁴. In comparison with other types of somatic cells, PBMNCs possess several advantages. The most significant advantage is their favorable gene expression patterns and epigenetic profiles. PBMNCs are short-lived in vivo and replenished frequently from activated hematopoietic stem cells, and may accumulate fewer mutations than skin fibroblasts do. Also, the way to sample PBMNCs is less invasive than that for fibroblasts, and it takes a shorter period of time to expand PBMNCs (around 14 days) versus fibroblasts (around 28 days)¹⁶^,²². After centrifugation using a density gradient medium, four density gradients will be formed from bottom to top; the bottom layer contains granulocytes and erythrocytes, the second lower layer contains the density gradient medium, the third layer contains MNCs, and the top layer contains plasma. The yield of PBMNCs normally varies between individuals, particularly those of different ages. Generally, younger people tend to have a greater number of PBMNCs than older people. With the described protocol, about 1.8−3.4 x 10⁷PBMNCs could be isolated from 15 mL of PB. Among PBMNCs, CD34⁺ hematopoietic stem cells are relatively prone to reprogramming, and MNC medium may enrich CD34⁺ hematopoietic stem cells to some extent. It is expected that an equal or greater number of visible cells cultured with MNC medium remain after 14 days of expansion. SeV, an RNA non-integrative virus, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells¹⁸^,¹⁹. Recombinant SeV encoding reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC, can be generated as a temperature-sensitive mutant, which could be removed easily at 39 °C²⁰. With this protocol, we derived 8-20 iNSC colonies by reprogramming PBMNCs from one healthy volunteer or patient using 15 mL of PB. Then researchers could select colonies with good morphologies for further passaging and line establishment. In the published report, iNSCs of passage numbers 10, 20, and 30 showed similar proliferation rates, and could be passaged more than 50 times, showing a good self-renewal and proliferative capacity¹¹. iNSCs could be characterized by differentiation assays and immunostaining for neural stem cell markers SOX2, PAX6, NESTIN and OLIG2, and the proliferative marker Ki67. iNSCs should express those neural stem cell markers and possess a differentiation ability to become TUJ1⁺, MAP2⁺ neurons (after 6 weeks), GFAP⁺ astrocytes (after 6 weeks) and OLIG2⁺, O1⁺ oligodendrocytes (after 7-8 weeks)¹¹. However, one limitation of this protocol is that MNC medium is preferentially favorable to erythroblast expansion, which may render it not suitable for generating iNSCs from patients who are deficient in erythroblast development. Besides, the efficiency of reprogramming PBMNCs to iNSCs is not high. One may enhance reprogramming efficiency by using certain small molecules or increasing the yield by using more starting PBMNCs²³.

The method about the differentiation of iNSCs into DA neurons described here builds upon a great number of protocols for neural differentiation. As PD is mainly caused by degeneration of DA neurons located in the midbrain¹^,², the aim of the protocols is focused on the derivation of specialized VM DA neurons, which arise from floor plate cells during development²⁴. Induction of VM floor plate cells with neurogenic potentials depends on two key morphogens, SHH and FGF8b⁶^,²⁵^,²⁶. SHH is a ventralizing morphogen, secreted by notochord²⁶^,²⁷. Here we replaced SHH with the small molecule SAG1, a SHH pathway agonist that is more economical than SHH. Also, basic neural culture medium and supplement (Table of Materials) are important for neuronal survival and differentiation. With this protocol, DA precursors were obtained towards the end of the first stage, and more mature DA neurons were generated at the end of the second stage. Increasing the period of time of the second stage to 40-55 days could further enhance the proportion of mature DA neurons in culture. Included in the second stage medium are the retinoic acid, BDNF, GDNF, TGF-βIII, DAPT and cAMP, which have been demonstrated to be able to promote DA neuron maturation and survival. To test the efficiency of iNSCs in differentiation into DA neurons, the differentiated cells were examined for expression of markers NURR1, FOXA2, GIRK2 and TH. In a previous study, at the end of stage I (day 10), 87.76% and 65.33% cells expressed NURR1 and FOXA2, respectively¹¹. At the end of stage II (day 24), the percentage of NURR1⁺ cells reached 95.58% and the proportion of FOXA2⁺ cells reached 77.33%¹¹. At this time point, 57.23% and 28.55% cells were positive for TH and GIRK2, respectively¹¹. Similar to what has been observed for iPSC differentiation towards DA neurons, a batch to batch/line to line variation for iNSC differentiation also exists, which is influenced by factors such as cell state, the activity of small molecules used, and the plasticity of stem cells from different persons. It is also noteworthy that a key determinant of the differentiation efficiency is cell density. A seeding density of 5 x 10³ cells per 12 mm glass coverslip in 24-well plates is recommended. In fact, iNSCs exhibit a good proliferative rate during differentiation stage I. The iNSCs seeded in one well of a 24-well plate would give rise to a sufficient number of DA precursors by differentiation day 10−13 for transplantation into one mouse (2 x 10⁵ for each mouse).

This protocol presents a method for the establishment of reproducible and stable unilateral 6-OHDA-lesioned PD mouse models. The extent of 6-OHDA lesion can be estimated by behavioral assessment that measures the contralateral rotations after injection of apomorphine. Also, the degree of 6-OHDA-induced lesion can be quantified by post-mortem immunofluorescent staining for TH in the SNpc. Another type of neurotoxin used in PD modeling is 1-methyl-4-phenyl-1,2,3,6-tertahydropyridine (MPTP), which also disrupts dopaminergic pathways. Compared to MPTP, 6-OHDA could be administered uni- or bilaterally. Also, the MPTP method is more sensitive to animal age, gender and strain and thus may show a higher degree of variation between the animals²⁸. The critical factors include selecting mice with matching weight, injecting freshly prepared 6-OHDA solution and performing surgery accurately and quickly.

The procedures of cell transplantation of DA precursors into the striatum of lesioned mice are basically the same as the procedures for generation of 6-OHDA-lesioned PD mouse models, except that 6-OHDA is replaced by DA precursors at the injection step. The key factor in this part is searching the optimal time window of DA cell differentiation for transplantation. It has been demonstrated that a higher degree of stemness correlates with a higher survival rate but a lower potential of specification into mature DA neurons¹¹. However, a more mature stage of neural cells are more vulnerable, and show a lower survival rate after transplantation¹¹. Therefore, finding a suitable time window that balances the ability of maturation and survival is of significance. In a previous study, we transplanted cells of differentiation day 10 and 13 into the striatum of immunodeficient SCID-beige mice¹¹. One month after engraftment, immunofluorescent staining results revealed that about 88.63% and 93.13% were TUJ1-positive for day 10 and day 13 groups, respectively, and some TH⁺cells (5.30%) were detected from day 13 group but few TH⁺ cells from day 10 group. Nevertheless, compared to day 13 cells, day 10 cells gave rise to a slightly higher overall survival rate¹¹. The results revealed that day 10 to day 13 is an optimal time window of cells for engraftment¹¹. In this protocol, we used a mixture of DA cells from differentiation day 10 and day 13 at a ratio of 1:7 for engraftment, which showed a good result of survival and differentiation¹¹. Using a mixture of cells from day 10 and day 13 was based on a hypothesis that relatively immature and mature neural cells, when put together, may support each other, reminiscent of the in vivo situation in mice where neural stem cells are surrounded by mature neurons.

This protocol presents the method of generating iNSCs from PBMNCs by SeV infection, differentiating iNSCs into DA neurons, and transplanting DA precursors into 6-OHDA-lesioned PD mouse models. Using this protocol, one can generate iNSCs with potentials not only for treatment of PD, but also of other neurodegenerative diseases. Since the iNSCs represent a primitive NSC stage, they can be specified into different region-specific neural cells, such as spinal cord neurons or motor neurons, which may be of promising utility for treatment of amyotrophic lateral sclerosis or spinal cord injury. Besides, iNSCs derived from familial disease patients offer a platform to study mechanisms underlying disease onset and development, and to conduct drug screening tests.

There is more than one way to obtain DA neural cells for transplantation studies. DA cells can also be generated from iPSCs or by direct conversion²⁹. Compared with iPSCs, iNSCs are inherent with reduced risk of tumor formation, a shorter period of reprogramming and line establishment, and lineage-committed plasticity - only able to differentiate into neurons and glia¹¹. Compared with direct conversion, differentiating DA precursors from iNSCs gives rise to a higher yield and efficiency³⁰.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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–year Plan (CIT & 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).

Materials

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
TGFβⅢ	Peprotech	100-36E	Transforming growth factor βⅢ
Transferrin	R&D Systems	2914-HT-100G
Triton X 100	baiaoshengke		Nonionic surfactant
Trypan blue	Gibco	T10282
Xylazine	Sigma-Aldrich	X1126

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Zheng, W., Chen, Z. Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies. J. Vis. Exp. (149), e59690, doi:10.3791/59690 (2019).