[methods in molecular biology] somatic stem cells volume 879 || assessing the potential clinical...

147

Shree Ram Singh (ed.), Somatic Stem Cells: Methods and Protocols, Methods in Molecular Biology, vol. 879,DOI 10.1007/978-1-61779-815-3_10, © Springer Science+Business Media, LLC 2012

Chapter 10

Assessing the Potential Clinical Utility of Transplantations of Neural and Mesenchymal Stem Cells for Treating Neurodegenerative Diseases

Laurent Lescaudron , C. Boyer , Virginie Bonnamain , K. D. Fink , X. Lévêque , J. Rossignol , V. Nerrière-Daguin , A. C. Malouet , F. Lelan , N. D. Dey , D. Michel-Monigadon , M. Lu , I. Neveu , S. von Hörsten , P. Naveilhan , and G. L. Dunbar

Abstract

Treatments for neurodegenerative diseases have little impact on the long-term patient health. However, cellular transplants of neuroblasts derived from the aborted embryonic brain tissue in animal models of neurodegenerative disorders and in patients have demonstrated survival and functionality in the brain. However, ethical and functional problems due to the use of this fetal tissue stopped most of the clinical trials. Therefore, new cell sources were needed, and scientists focused on neural (NSCs) and mesenchymal stem cells (MSCs). When transplanted in the brain of animals with Parkinson’s or Huntington’s disease, NSCs and MSCs were able to induce partial functional recovery by promoting neuroprotection and immunomodulation. MSCs are more readily accessible than NSCs due to sources such as the bone marrow. However, MSCs are not capable of differentiating into neurons in vivo where NSCs are. Thus, transplanta-tion of NSCs and MSCs is interesting for brain regenerative medicine. In this chapter, we detail the methods for NSCs and MSCs isolation as well as the transplantation procedures used to treat rodent models of neurodegenerative damage.

Key words: Neural stem cells , Mesenchymal stem cells , Stem cell isolation , Brain transplantation

Current treatments for neurodegenerative diseases such as Parkinson’s (PD) and Huntington’s (HD) are limited and have little impact on the long-term health of the patient. However, transplants of neuroblasts derived from embryonic/fetal gangli-onic eminence (for use in HD) of ventral mesencephalon (for use

1. Introduction

148 L. Lescaudron et al.

in PD) have shown to survive and be therapeutically bene fi cial in rodent models of neurodegenerative diseases. Such studies have linked neuronal replacement and partial reinnervation of the denervated striatum after fetal cell transplantation to functional recovery. As such, neuroblast transplantation therapy may offer a viable treatment strategy for patients with these diseases. However, because of the lack of availability and ethical issues surrounding the use of human fetal or embryonic tissue, as well as the need to use immunosuppressors (which can damage kidney function), the clinical utility of this form of therapy for neurodegenerative diseases is severely limited. For example, some trials with PD patients receiving immunosuppression following transplanta-tion of neuroblasts result in debilitating dyskinesia, leading to a suspension of most of the clinical trials on neuroblast transplan-tation ( 1, 2 ) .

More recently, researchers have focused on new cell types such as adult neural (NSCs) and mesenchymal (MSCs) stem cells. When transplanted as neurospheres in the brain of animal models of PD or HD, NSCs were able to induce some functional recovery ( 3, 4 ) by promoting neuroprotection ( 5 ) and immunomodulation ( 6, 7 ) . However, NSCs are dif fi cult to obtain, as they are located deep inside the adult brain or in various regions of the fetal brain. As an alternative, MSCs, which are more easily accessible and have been shown to restore functional de fi cits ( 8– 10 ) in rodent models of neurological disease, have gained increasing attention as a poten-tial therapeutic approach. Unlike NSCs, transplanted MSCs do not give rise to neurons, but have important immunosuppressive prop-erties ( 11 ) beyond what is observed in transplanted neuroblasts ( 12 ) . In addition, MSCs provide growth support, such as brain-derived neurotrophic factor, and produce extracellular matrix pro-teins, such as collagen type I, and fi bronectin, which support cell survival and function ( 10 ) .

Thus, transplantation of MSCs derived from the marrow is a promising alternative to transplantation of neuroblasts and NSCs, primarily due to their ability to provide trophic support to injured/neurodegenerative neurons, as well as their capacity to delay or halt degenerative processes.

Presently, our labs have been engaged in collaborative proj-ects testing the ef fi cacy of transplanting MSCs and/or NSCs in various models toxic and transgenic of HD and PD ( 8– 11, 13, 14 ) . The following protocols have proven to be successful and could provide useful paradigms for further studies using trans-plants of MSCs and NSCs in animal models of similar neurode-generative diseases.

14910 Assessing the Potential Clinical Utility of Transplantations…

1. Sprague-Dawley embryos at 15 days of embryonic life. 2. Binocular dissecting microscope (Nikon). 3. Hanks’ Balanced Salt Solution (HBSS) without phenol red

(Sigma-Aldrich) supplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Gibco).

4. Basal medium: Dulbecco’s Modi fi ed Eagle’s Medium (DMEM)/Ham’s F12 1:1 (Gibco) supplemented with 33 mM D -glucose, 5 mM HEPES (pH 7.2), 100 U/mL.

5. Penicillin and 0.1 mg/mL streptomycin, 2 mM L -glutamine. 6. Complete medium: basal medium with 10% heat-inactivated

fetal calf serum (FBS or FCS, Lonza). 7. De fi ned medium: basal medium supplemented with N2

supplement (Gibco). 8. Phosphate-buffered saline (PBS): prepare 10× stock with 1.37 M

NaCl, 27 mM KCl. 9. 100 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 (adjust to pH 7.4 with

HCl if necessary). Prepare working solution by dilution of one part with nine parts of distilled water and autoclave before storage at room temperature.

10. Dissolve trypsin TPCK treated from bovine pancreas (Sigma-Aldrich) in PBS at 25 mg/mL, and deoxyribonuclease I from bovine pancreas (Sigma-Aldrich) is dissolved in HBSS at 10 mg/mL. Both are stored in aliquots at −20°C and then used for tissue dissociation as required.

11. Human basic fi broblast growth factor (bFGF; PeproTech EC, London) is dissolved at 25 m g/mL in PBS supplemented with 4% bovine serum albumin (BSA). bFGF is stored in aliquots at −20°C and then added to cell culture dishes as required.

12. Fetal bovine serum albumin (BSA; Sigma-Aldrich). 13. Poly- L -ornithine (Sigma-Aldrich). 14. Sterile 70- m m fi lter (BD Biosciences). 15. P1000 Pipetman (Thermo Fisher Scienti fi c). 16. 15-mL Falcon tube (Thermo Fisher Scienti fi c).

1. Binocular dissecting microscope (Nikon). 2. A vertical laminar fl ow hood. 3. Rat brain atlas.

2. Materials

2.1. Isolation and Culture of Neural Stem Cells

2.1.1. From Fetus NSCs

2.1.2. From Adult Neural Stem Cells


4. HBSS (Sigma-Aldrich). 5. 0.25% EDTA/trypsin solution (Sigma-Aldrich). 6. Fetal bovine serum (Gibco). 7. DNase I (Sigma-Aldrich). 8. Poly- L -ornithine-coated dishes (50 mg/mL; Sigma-Aldrich). 9. De fi ned medium: DMEM/Ham’s F12 (1:1, v/v; Sigma-

Aldrich), 33 mM glucose (Sigma), 5 mM HEPES (Sigma-Aldrich; pH 7.2), 5 mg/mL streptomycin, and 5 U/mL penicillin (Sigma-Aldrich) supplemented with 10% FBS or FCS.

10. B27 supplement (Gibco). 11. Epidermal growth factor (EGF) (Invitrogen). 12. Fibroblast growth factor-2 (bFGF, Invitrogen). 13. Poly- L -ornithine (Sigma-Aldrich). 14. 10-cm Petri dish (Nunc, Thermo Fisher Scienti fi c). 15. 75-cm² fl ask (Nunc, Thermo Fisher Scienti fi c). 16. Fire-polished Pasteur pipettes (Thermo Fisher Scienti fi c).

1. Sprague-Dawley rat (2–3 months old). 2. Anesthesia : 0.4% Rompun (Bayer, 4%) with 4% kétamine

(Panpharma, 5%). Inject 1.3 mL/kg of the mixture (IM). 3. Alpha Modi fi ed Eagle’s Medium ( a MEM, Gibco). 4. Fetal calf serum (FCS; PAA, the Cell Culture Company). 5. 0.25% Trypsin-EDTA solution (Gibco). 6. Antibiotics: penicillin/streptomycin (Gibco). 7. Hemocytometer (Thermo Fisher Scienti fi c). 8. Culture fl ask (75/150 cm 2 Nunc, Thermo Fisher Scienti fi c). 9. 25-Gauge syringe (Thermo Fisher Scienti fi c).

1. FACSCalibur fl ow cytometer (Becton, Dickinson and Company).

2. 0.1 M PBS (Sigma-Aldrich). 3. Bovine serum albumin (BSA; Sigma-Aldrich). 4. Sodium azide (Sigma-Aldrich). 5. PBN: 500 mL of 0.1 M PBS + 1% BSA + 0.1% azide.

1. Stereotaxic frame (Stoelting). 2. Ten microliter Hamilton syringe. 3. Automated microinjector (Phymep). 4. A drill (Foredom C094369, Phymep). 5. Bone wax (Ethicon LLC).

2.2. Isolation and Culture of Mesenchymal Stem Cell

2.3. Preparation of MSCs for FACS Analysis (Optional)

2.4. Cell Transplantation


6. Sterile wound clips (World Precision Instrument) or sterile sutures (Premocron).

7. PKH-26 kit (Sigma-Aldrich). 8. Hoechst 33258 (Sigma-Aldrich). 9. Eosine (Sigma-Aldrich). 10. Trypan blue (Sigma-Aldrich).

1. 100 U of hyaluronidase (Sigma-Aldrich). 2. Sterile 0.1 M PBS. 3. Rompun (Panpharma). 4. Ketamine (Panpharma). 5. PKH-26 kit (Sigma-Aldrich). 6. P20 Gilson pipette (Thermo Fisher Scienti fi c).

1. pMSCVpuro Vector (Clontech). 2. RetroPack PT67 packaging cells (Clontech). 3. DMEM (Sigma-Aldrich). 4. DMEM, containing 90% DMEM, 10% fetal bovine serum

(FBS) or fetal calf serum (FCS), 4 mM L -glutamine, 100 U/mL penicillin, and 100 m g/mL streptomycin.

5. Isocove Modi fi ed Dulbecco Medium (IMDM) supplemented with 9% FBS or FCS, 9% horse serum (HS), 100 m g/mL peni-cillin, 100 U/mL streptomycin, and 12 m M L -glutamine (Sigma-Aldrich).

6. FuGENE HD Transfection Reagent (Roche). 7. Puromycin (Sigma-Aldrich). 8. 0.45- m m cellulose acetate fi lter (Thermo Fisher Scienti fi c). 9. Polybrene (Sigma-Aldrich).

1. Anesthesia: 0.4% Rompun (Bayer, Rompun 4%) with 4% kétamine (Panpharma, ketamine 50 mg/mL). Inject 2.6 mL/kg of the mixture (IP).

2. PBS or 0.9% saline at 4°C. 3. 4% Paraformaldehyde (PAF) in 0.1 M in phosphate buffer (PB)

at 4°C. 4. Peristaltic pump (Cole-Palmer Master fl ex Pump Drive easy-

load model 7518-10) and 2.5-mm-diameter tubing. 5. Blunted cannula (Master fl ex, 16 gauge). 6. Cryoprotectant solution: 15 and 30% sucrose (Sigma-Aldrich)

in 0.1 M PB.

2.5. Nasal Administration of MSCs ( 15 )

2.6. Transfection of MSCs

2.7. Perfusion of the Animals


1. Rat embryos at E15 were collected by hysterectomy. 2. Isolate the whole brain. 3. Cut from the mesencephalon to the frontal cortex, above

the eyes. 4. Remove meninges. 5. Collect brain tissues into a 15-mL Falcon tube containing

2 mL of ice-cold HBSS. 6. Continue under sterile laminar fl ow hood and use strict sterile

technique. Put the brain tissue into a culture dish and use a scalpel blade to mince tissue for 30 s.

7. Using 1,000- m L plastic tips together with a P1000 Pipetman, transfer the minced embryonic tissue within a total volume of 5 mL of basal medium into a 50-mL Falcon tube.

8. Add 0.1 mg/mL of trypsin and incubate for 15 min in a 37°C water bath.

9. Return the tube to the hood and add 10 mL of complete medium (containing 10% FBS or FCS) to inhibit the enzy-matic process. Let the tube at room temperature for 5 min.

10. Add 0.1 mg/mL of DNase I and incubate the tube for 10 min in a 37°C water bath.

11. Mechanically dissociate the tissue with a 5-mL pipette and avoid air bubbles.

12. Let the suspension settle for 5 min and transfer 10 mL of the cell suspension to a clean, labeled tube, leaving 5 mL behind. To the latter, triturate again ten times with a P1000. Let the suspension settle for 5 min. Transfer all but 200 m L from this tube to the labeled tube; thus, pool the cells from both tritura-tion steps.

13. Pellet the cells by centrifugation at 50 × g for 10 min at room temperature.

14. Remove the supernatant. 15. Gently resuspend the pellet in 1 mL of complete medium. 16. Plate the cells in 100 × 20-mm culture dishes containing 10 mL

of complete medium in order to have the quantity of cells cor-responding to fi ve brains per dish.

17. Incubate at 37°C, 5% CO 2 in a humidi fi ed incubator for 12 h. 18. After 12 h in complete medium, transfer culture dishes to the

hood. Using a 10-mL pipette, aspirate half of the medium and rinse two times to remove all the remaining cells.

3. Methods

3.1. Isolation and Culture of Neural Stem Cells

3.1.1. Fetal NSCs


19. Collect the medium containing the fl oating cells into a 50-mL tube and pellet the cells by centrifugation at 50 × g for 10 min at room temperature.

20. Remove the supernatant and gently resuspend the cells into fresh de fi ned medium.

21. Plate the cells into 100 × 20-mm culture dishes in order to have the quantity of cells corresponding to one brain per dish (1:5 split) and incubate in 10 mL of fresh de fi ned medium.

22. Add bFGF (25 ng/mL). 23. Incubate at 37°C, 5% CO 2 in a humidi fi ed incubator. 24. Add bFGF every 2 days to stimulate the proliferation of NSPC

as spherical clusters. These primary neurospheres should be ready for subculture 5 days after initial plating.

25. At day 5, bring culture dishes to the hood. Collect the medium containing the neurospheres and transfer it to a 50-mL Falcon tube, removing the cells by rinsing each culture dish.

26. Pellet the cells by centrifugation at 50 × g for 10 min at RT and resuspend the neurospheres in 1 mL of fresh de fi ned medium. Dissociate mechanically the neurospheres by gently pipetting up and down in order to get a homogenous cell suspension.

27. Plate the cells in new 100 × 20-mm culture dishes (1:2 split). Add bFGF as required and incubate in 10 mL of fresh de fi ned medium.

28. Incubate at 37°C, 5% CO 2 in a humidi fi ed incubator for another 5 days to allow the formation of secondary neurospheres.

29. Add bFGF at day 7. 30. At day 10, fl oating neurospheres are collected in a 50-mL

Falcon tube and centrifugated at 50 × g for 10 min at RT. 31. Gently resuspend the pellet in complete medium in order to

have 200 neurospheres/mL (see Fig. 1a ). 32. Plate 1 mL of neurospheres onto poly- L -ornithine (PORN)-

coated cover slips in a 12-well plate. 33. Incubate at 37°C overnight to allow cells to adhere to the

cover slips. 34. Replace the complete medium by 1 mL of de fi ned medium/

well. 35. Allow neurospheres to differentiate for 7 days at 37°C. 36. Fix neurospheres by adding 4% paraformaldehyde in PBS for

15 min at RT and then wash the cells three times with PBS. 37. Incubate the cells for 1 h at RT in a permeabilizing/blocking

solution containing 4% BSA, 0.1% Triton X-100, and 10% nor-mal goat serum in 1× PBS (PBT-NGS).


38. Add 300 m L/well of anti-nestin antibody diluted in PBT (rat 401; 1/1,000; Developmental Studies Hybridoma Bank, Iowa City, IA) and incubate overnight at 4°C.

39. Wash the cells three times with PBS. 40. Incubate cells with 300 m L/well of FITC-conjugated goat

anti-mouse IgG diluted in PBT (1/250; Jackson Immu-noResearch, Cambridgeshire, UK) for 2 h at RT in the dark.

41. Wash the cells three times with PBS and mount the cover slips on slides using DABCO antifading medium.

42. Analyze the slides by fl uorescence microscopy (see Fig. 1b ).

1. Under a binocular dissecting microscope, dissect out 1-mm 3 tissue around the left and the right ventricles of the rat (between +0.5 and +1 mm anteroposterior level, as compared to bregma level) on a bench or in a horizontal laminar fl ow hood (Fig. 2f ).

2. Transfer in a 10-cm Petri dish fi lled with 10 mL HBSS the 1-mm 3 tissue.

3. Mechanically dissociated the tissue in a 0.25% EDTA/trypsin solution (Sigma) under sterile conditions in a vertical laminar fl ow hood.

4. After 10 min, deactivate the trypsin with 2 mL of FBS or FCS (see Note 1).

3.1.2. Adult NSCs

Fig. 1. Fetal neural stem cells (fNSCs). In vitro ( a ), in proliferating conditions (+ bFGF no coating, no FCS), NSCs isolated from fetal brain are proliferating as a neurosphere. ( b ) In differentiating conditions (+ FCS, + coating, no bFGF), the neuro-sphere attaches to the bottom plates, and the NSCs undertake their differentiation, as evidenced by cell process extension. As visualized in green using nestin antibodies, these neural progenitors still express this intermediate fi lament considered as a marker for neuroepithelial stem cells. In vivo ( c ), NSCs isolated from rat fetal brain and transplanted into the striatum (St) of an adult rat survive and differentiate. The micrograph shows fNSC transplant (NSC-T) inside the striatal parenchyma (St) of an eGFP transgenic rat ( green ).


5. Expose the cell preparation to 10 mg/mL of DNase I for 10 min at 37°C and triturate with fi re-polished Pasteur pipettes.

6. Removal of aggregates by decantation.

Fig. 2. Adult neural stem cells (aNSCs) and mesenchymal stem cells (MSCs). In vitro, aNSC grown as neurospheres and plated onto poly- L -ornithine glass cover slips showed positive labeling for nestin (( a ) labeling neural progenitors) and NeuN antibodies (( b ) labeling neuron nucleus). Mesenchymal stem cells (MSCs) at passage 5 ( c ) were labeled with Dapi ( blue ; labeling the cell nucleus) and CD90 antibodies ( green ; labeling the cell body). In vivo, aNSCs (labeled with PKH-26 ) and MSCs (labeled with Hoechst ), respectively, were transplanted into the striatum of Huntington’s disease transgenic rats ( 13 ) at 12 months of age. ( d ) Five months after transplantation, the PKH-26-labeled aNSCs (in red ) were present inside the rat striatum but did not differentiate into GABAergic neurons (Darpp-32 in green ). ( e ) Hoechst-labeled MSCs were also observed within the striatum after transplantation. A few of them were positive for NeuN ( red ), but none of them differenti-ated into GABAergic neurons (Darpp-32 in green ). ( f ) In vivo labeling of aNSCs using Bromodeoxyuridine (BrdU). Since proliferating cells incorporate BrdU into their nucleus, aNSCs could be localized in the brain using this synthetic nucleoside. The micrograph shows the presence of aNSCs (in brown ) in the right SVZ. The right and left SVZs are currently dissected out to get aNSCs for transplantation. St striatum; V lateral ventricle. ( g ) Characterization of rat MSCs by FACS. At passage 1, 95% of the MSCs were positive for CD 90.


7. Collect the cells by centrifugation (50 × g for 10 min at RT) and plate onto poly- L -ornithine-coated dishes (50 mg/mL; Sigma) at a fi nal density of 10 5 cells/cm 2 .

8. Plate the cells in a mixture of DMEM/Ham’s F12 (1:1, v/v), 33 mM glucose, 5 mM HEPES (pH 7.2), 5 mg/mL strepto-mycin, and 5 U/mL penicillin supplemented with 10% FBS or FCS.

9. Twelve hours after plating, place the fl oating cells in a 75-cm² fl ask in de fi ned medium composed of DMEM/Ham’s F12 (1:1, v/v), 33 mM glucose, 5 mM HEPES (pH 7.2), 5 mg/mL streptomycin, and 5 UI/mL penicillin (Sigma) supple-mented with B27 supplement (Gibco), 10 ng/mL EGF, and 100 ng/mL basic fi broblast growth factor-2 (FGF-2).

10. Grow NSCs until fl oating neurospheres reached con fl uency. 11. Then dissociate the neurospheres using 0.25% EDTA/trypsin

solution (see Note 1). 12. After 5 min, inhibit the trypsin by adding the same volume of

FBS or FCS to the fl ask. 13. Centrifuge at 50 × g for 10 min. 14. Plate the pellet in a new fl ask fi lled with de fi ned medium to

allow the formation of secondary neurospheres (see Fig. 2a , b).

1. Sprague-Dawley rats were sacri fi ced in the institute’s accred-ited animal facility in accordance with the institutional guide-lines of the Institut National de la Santé et de la Recherche Médicale (INSERM).

2. Under a sterile hood, extract the bone marrow cells (BMC) from femoral and tibia bones by aspiration with a 25-gauge syringe and put into a sterile 15-mL tube fi lled with 5 mL of the a MEM solution containing the Alpha Modi fi ed Eagle’s Medium ( a MEM), 20% FBS or FCS, 100 U/mL penicillin, and 100 m g/mL streptomycin.

3. After several aspirations, fl ush the bone canal by a MEM solu-tion in order to remove all the BMC.

4. Following extraction, suspend the BMC in 10 mL of a MEM solution.

5. After quanti fi cation using a hemocytometer, for this fi rst plat-ing, use 75-cm 2 fl ask and put 0.6 million cells per fl ask (in 20 mL a MEM solution) at 37°C/5% CO 2 in the incubator for no more than 24 h.

6. Mesenchymal stem cells (MSCs) are selected by their proper-ties to attach to the plastic fl ask. After one night in the incubator at 37°C, change the a MEM solution in order to remove the nonattached cells and add the same volume of 37°C a MEM solution (see Fig. 2c ).

3.2. Isolation and Culture of Mesenchymal Stem Cell


7. Let the fl ask at 37°C, 5% CO 2 in a humidi fi ed incubator for 8–10 days without medium change.

8. When the cells reached 80/90% of con fl uence (8–10 days), remove the supernatant.

9. Wash twice the fl ask with 37°C a MEM (without FBS or FCS) in order to remove nonadherent cells and remaining a MEM solution (i.e., the FBS or the FCS). Remove supernatant.

10. Put 4 mL of a 37°C 0.25% trypsin-EDTA solution into the 75-cm 2 fl ask and, immediately after, put the fl ask back into the incubator for 5 min. Shake the fl ask after 3 min. After 5 min, shake the fl ask again and take it out of the incubator. Depending on the culture conditions, one can check on inverted micro-scope to see if cells are detached from the bottom of the fl ask after 4/5 min. If not, put fl ask back at 37°C for additional 1 min and check again. Usually, 5 min of trypsin-EDTA solu-tion is long enough (see Note 1).

11. Following detachment of cells, inhibit the trypsin by adding 20 mL of a MEM solution or 4 mL of FBS or FCS. Shake the fl ask.

12. Centrifuge the cells at 100 × g for 10 min at 4°C. 13. Resuspend the pellet (6,000 cells/cm 2 ) into the a MEM solu-

tion (20 mL) in a 75-cm² fl ask. 14. About 3/4 days later (85% of con fl uence), trypsinize the cells

(as previously described in step 11 which will be the second passage; see Note 1).

15. Then, plate the cells as previously describe in step 14 into 75-cm 2 fl ask (8,000 cells/cm 2 ) containing 20 mL medium.

16. After 3/4 days (85% of con fl uence), repeat 8–14 (third passage). 17. After 3/4 days (85% of con fl uence), repeat 8–14 (fourth

passage). The MSCs are then ready for transplantation (see Note 2).

1. Plate 250,000–500,000 MSCs per well into 96-well plate (v- or round-bottom).

2. Complete each well to 200 m L with PBN. 3. Centrifuge at 200 × g for 1 min. 4. Check to make sure there is a pellet at the bottom of your well

plate and remove the supernatant. 5. Add 30 m L primary antibody per well for 30 min on ice (1/500

dilution in PBN). 6. Centrifuge at 200 × g for 1 min. 7. Remove the supernatant. 8. Add 100 m L of PBN.

3.3. MSCs Preparation for Cell Charac-terization by FACS (Optional)


9. Centrifuge at 200 × g for 1 min. 10. Remove the supernatant. Add 100 m L of PBN. Remove PBN. 11. Add 30 m L secondary antibody per well for 30 min on ice

(1/300 dilution in PBN). 12. Centrifuge at 200 × g for 1 min. Remove the supernatant. Add

100 m L of PBN. 13. Store at 4°C and use within 48 h. 14. Use primary antibodies (At least 90% of the cells must be CD90

positive). 15. Use secondary antibodies (see Fig. 2g ).

1. Staining the cells just prior transplantation is useful to localize the transplanted cells later on brain sections.

2. The day of the transplantation, add to the MSCs culture medium contained in the culture fl ask some Hoechst 33258 (5 m g/mL, Sigma) for 5 min at 37°C. The Hoechst labels in blue the nucleus of the cell ( UV excitation fl uorescence fi lter). For Hoechst 33258 staining, wash the fl ask containing the MSCs (3 × 5 min in a MEM) at 37°C in order to remove the excess of Hoechst 33258 staining.

3. Trypsinize the cells as previously described (see Note 1). 4. For PKH-26 staining (5 m L of PKH-26 for six millions of MSCs),

the MSCs must be in suspension. PKH-26 labels the plasmic cell membrane in red (red excitation fl uorescence fi lter).

5. Put 4 mL of a 37°C 0.25% trypsin-EDTA solution into the MSC 75-cm 2 fl ask and, immediately after, put the fl ask back into the incubator for 5 min. Shake fl ask after 3 min. After 5 min, shake the fl ask again and take it off the incubator. If needed, mostly for NSCs, mechanically dissociate the tissue with a 5-mL pipette and avoid air bubbles (see Note 1).

6. After 5 min of incubation at 37°C, inhibit trypsin by adding 20 mL of a MEM solution or 4 mL of FBS or FCS. Shake the fl ask.

7. Centrifuge the cells at 100 × g for 10 min at 20°C. 8. Resuspend the pellet into the 1 mL of a MEM without FNS

or FCS. 9. After quanti fi cation using a hemocytometer, add 45 mL

a MEM without FBS or FCS. 10. Centrifuge the cells at 100 × g for 10 min at 20°C. 11. Resuspend the pellet into 250 m L of diluent C from the

PKH-26 kit. 12. In another 50-mL Falcon tube, put 5 m L of PKH-26 into

another 250 m L of diluent C.

3.4. Transplantation of MSCs and NSCs into the Rat Striatum

3.4.1. Preparation of Cell Suspension


13. Add the 250 m L of diluent C with the cells (n°11) into the 5 m L of PKH-26+ 250 m L of diluents C solution (n°12). Final volume: 505 m L.

14. Shake with hand the 505 m L in the dark for 5 min. 15. Add 505 m L of FBS or FCS to stop the reaction. Final volume:

1.010 mL. 16. Shake with hand for 1 min. 17. Add 1 mL of a MEM with FBS or FCS. Final volume:

2.010 mL. 18. Centrifuge the cells at 100 × g for 7 min at 20°C. 19. Resuspend the pellet into a MEM with FCS. 20. Redo 18 and 19 twice (total of washes: 3; for the last wash, use

sterile PBS) 21. Suspend the cells in PBS (200,000 cells/ m L). 22. For neurospheres, dissociate the neurospheres with gentle pas-

sages through P20 Gilson and perform the PK-26 staining as for the MSCs.

23. Put the Eppendorf tube containing the cell suspension at the needed concentration (200,000 cells/ m L) on the ice.

24. Control for the viability of the transplanted cells with 0.15% eosin solution. Dilute 1:10 the cells in 0.15% eosin solution and count the red cells using the hemocytometer. Cells becom-ing red are dying cells. Another solution to estimate the viabil-ity of the transplanted cells is to use some 0.4%, trypan blue solution. Add an equal volume of trypan blue solution to the cell suspension tested and mix by gentle pipetting. In this case, dead cells appear in blue. It is worth to do viability tests prior the fi rst transplantation and after the last transplantation (see Notes 3 and 4).

1. Anesthetize adult rat by intramuscular injection of Rompun/ketamine (1.33 mL/kg). The animals can also be sedated with iso fl urane gas and O 2 using the Tem SEGA TEC III evapora-tor (Sega Electronique, Lormont, France).

2. Shave the hair of the rat at the site of surgery. 3. Clean the area with Betadine (Mediapharma, or chlorhexidine)

(4%; Molnlycke Health Care) then with 70% alcohol. 4. Put some mineral oil on the rat eyes to prevent retinal damage

during surgery due to surgical lights. 5. Place animal in a stereotaxic frame with the incisor bar set at

−3.3 mm above the level of the ear bars. A 2% lidocaine (Pharmacal) gel can be placed on the tips of the ear bar prior to placement in order to prevent any additional pain.

3.5. Transplantation of NSCs and MSCs (Fig. 2d , e)


6. After incision over the scalp and retracting the skin, position the needle of a Hamilton syringe (not yet containing the cell suspension) mounted on an automated microinjector on the bregma which is used as reference point.

7. Then, using a drill, two burr holes (0.5 mm) are performed over each hemispheres (for bilateral transplantation), directly over the striatum (coordinates, relative to bregma: +0.5 mm AP; ±2.6 mm ML; −6 and −5 mm DV with incisor bar at −3.3 mm; according to the rat brain atlas).

8. Expose the dura mater. If some bleeding occurs, stop it by using the extremity of a cotton swab previously soaked in a 4°C sterile 0.9% saline solution.

9. Fill the Hamilton syringe with 3 m L of cell suspension. Do not remove the Hamilton syringe from the from the stereotaxic frame.

10. At each transplantation site (i.e., bilateral transplantation), deliver two injections of 1 m L of cell suspension with a 10- m L Hamilton syringe (0.35–0.8 m L/min) mounted on an auto-mated microinjector. A total of 400,000 cells are injected in each striatum. In the case of co-transplantation (i.e., MSCs + NSCs), 200,000 MSCs and 200,000 NSCs are mixed in the same cell suspension solution and injected together (see Notes 3 and 4).

11. Wait 5 min before withdrawing slowly (4 min) the syringe. 12. The burr hole can be sealed with bone wax, but not necessary

if no more than 1 mm of diameter. 13. Put back on place muscle layers and scalp. 14. The skin is sutured using sterile wound clips (9 mm) or sterile

sutures. 15. Put back the animal on his side in a resting cage. 16. Put back the animal in its home cage when awake and moving

freely. 17. Postoperative monitoring is undertaken to assure that animal is

not in distress. The incision site is observed for redness, swell-ing, drainage, and odor.

18. Monitor the weight of the animal as a loss would indicate some animal distress.

1. Prepare a solution of 100 U of hyaluronidase dissolved in 5 m L of sterile PBS.

2. Label the MSCs as in 3.4.1. 3. Prepare the MSC suspension (300,000 MSCs in 24 m L sterile

PBS) and put the aliquot containing the cell on ice.

3.6. Nasal Administration of MSCs ( ( 15 ) and Fig. 3 )


4. Anesthetize adult rat by intramuscular injection of Rompun/ketamine (1.6 mL/kg).

5. Put the animal on his back. 6. Administrate into one of the two nostrils, 5 m L of hyaluroni-

dase solution with a P20 Gilson pipette. 7. Thirty minutes after the hyaluronidase treatment, shake the

aliquot containing the cells and inject MSC suspension into the same nostril (every 2 min, inject 6 m L of the MSC suspen-sion using a P20 Gilson pipette).

8. Put back the animal on his side in a resting cage. 9. Put back the animal in its home cage when awake and moving

freely. 10. Usually, no postoperative follow-up 24 h after surgery is needed.

Transfection of MSCs with gene/pMSCVpuro was used to improve the secretion of BDNF and EGF by MSCs ( 9 ) .

1. 12–24 h before transfection, plate RetroPack PT67 cells at 60–80% con fl uency with DMEM culture medium containing 90% DMEM (Sigma), 10% FBS or FCS (Gibco), 4 mM L -glutamine, 100 m g/mL penicillin, and 100 m g/mL strepto-mycin (Sigma).

2. Transfect the puri fi ed gene/pMSCV plasmid DNA (Clontech) into the packaging cells using any standard transfection protocol.

3. 36 h posttransfection, change the medium with a selection medium: add puromycin at 10 m g/mL. After the cells are

3.7. Transfection of Mesenchymal Stem Cells

Fig. 3. Nasal administration of MSCs in a hemiparkinsonian rat. (1) Unlesioned substantia nigra showing tyrosine hydroxylase (TH)-positive neurons. (2a) Lesioned substantia nigra showing the loss of TH-positive neurons. (2b) Nostril-administrated MSCs were able to reach the damage substantia nigra. The same brain sections are shown in H2a and H2b. In H2b, the red fl uorescent PKH-26-labeled MSCs are visible in the area where TH-positive neurons were lost.


cultured for 7–10 days, isolate large, healthy colonies as stable virus-producing cell lines.

4. Produce virus from the stable virus-producing clone: plate the selected clone at 60–80% con fl uency to the desired culture vol-ume. Viral supernatant is harvested in 24-h intervals until the cells are no longer viable.

5. (Optional) Storage of viral stocks: centrifuge the supernatant at 500 × g for 10 min. Aliquot cleared supernatant into single use tubes. Store tubes at −70°C.

6. 12–18 h before infection, plate the target MSC cells in IMDM medium at a cell density of 1–2 × 10 5 /60-mm plate to obtain a con fl uency of 40–60% at the infection time.

7. Collect medium from the infected packaging cells, or from the viral stock, and fi lter medium through a 0.45- m m cellulose acetate fi lter.

8. Change the culture medium of the target cells with the fi ltered viral medium supplemented with polybrene at a fi nal concen-tration of 4–8 m g/mL.

9. Replace medium with fresh medium after 24 h of incubation. To increase infection ef fi ciency, a second time infection may be carried out 12–24 h after the initial infection.

10. 48 h after incubation, replace the medium with fresh medium containing 10–15 m g/mL puromycin.

11. Subject cells to puromycin selection for 1–2 weeks, changing medium as necessary.

12. The transfected individual clones grow large and look healthy. 13. Real-time PCR can be used to verify the integrated gene copy

numbers and Western blotting to con fi rm the gene expression.

1. Trypsin, which is harmful to cells, must be neutralized with fetal calf serum or fetal bovine serum in order to stop trypsin activity.

2. Reports have shown that the properties of rat MSC change with passages and their survival in the rat brain parenchyma has been greatly increased when passaged 4/5 times ( 11 ) compared to higher passages.

3. It is important to quantify MSCs and NSCs viability before the fi rst transplantation procedure using eosine or trypan blue solutions. When labeled by PKH-26, MSCs viability is 95% for the fi rst transplantation and about 85% 8 h later. If MSCs are

4. Notes


not labeled or labeled with Hoechst, 95% viability is observed for 12 h. NSCs viability is always lower than MSCs viability, so it is advised to prepare a fresh suspension of NSCs every fi ve transplantation procedures.

4. Unlabeled or labeled cell suspensions (put in the dark) must remain in an Eppendorf tube on a bed of ice in order to increase viability until the transplantation.

Acknowledgments

The nestin monoclonal antibody developed by Susan Hock fi eld was obtained from the Developmental Studies Hybridoma Bank. This work was supported by the “Association Française contre les Myopathies” (AFM-France), the “Fédération des Groupements de Parkinsoniens-CECAP-France,” L’Association Huntington France, and Progreffe Foundation (INSERM U643).

D. Michel-Monigadon and F. Lelan were supported during their Ph.D. thesis by INSERM/Région Pays de la Loire. V. Bonnamain and Julien Rossignol were supported during their Ph.D. thesis by the Ministère de l’Enseignement Supérieur et de la Recherche and from Progreffe Foundation.

Xavier Lévêque was supported by the University of Nantes during his postdoctoral stay. Gary Dunbar was supported by the Field Neurosciences Institute and John G. Kulhavi Professorship. All members of CMU were also supported by the Field Neurosciences Institute.

References

1. Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W, Kao R, Dillon S, Win fi eld H, Culver S, Trojanowski JQ, Eidelberg D, Fahn S (2001) Transplantation of embryonic dop-amine neurons for severe Parkinson’s disease. N Engl J Med 344:710–719

2. Hagell P, Piccini P, Bjorklund A, Brundin P, Rehncrona S, Widner H, Crabb L, Pavese N, Oertel WH, Quinn N, Brooks DJ, Lindvall O (2002) Dyskinesias following neural transplan-tation in Parkinson’s disease. Nat Neurosci 5:627–628

3. Svendsen CN, Smith AG (1999) New prospects for human stem-cell therapy in the nervous sys-tem. Trends Neurosci 22:357–364

4. Vazey EM, Chen K, Hughes SM, Connor B (2006) Transplanted adult neural progenitor cells survive, differentiate and reduce motor function impairment in a rodent model of

Huntington’s disease. Exp Neurol 199:384–396

5. Michel-Monigadon D, Bonnamain V, Nerrière-Daguin V, Dugast AS, Lévèque X, Plat M, Venturi E, Brachet P, Anegon I, Vanhove B, Neveu I, Naveilhan P (2011) Trophic and immunoregulatory properties of neural precur-sor cells: bene fi t for intracerebral transplanta-tion. Exp Neurol 230:35–47

6. Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L, Salani G, Martinello M, Cattalini A, Bergami A, Furlan R, Comi G, Constantin G, Martino G (2005) Neurosphere-derived multipotent precursors promote neuroprotec-tion by an immunomodulatory mechanism. Nature 436:266–271

7. Bonnamain V, Neveu I, Naveilhan P (2011) In vitro analyses of the immunosuppressive properties of neural stem/progenitor cells using


anti-CD3/CD28-activated T cells. Methods Mol Biol 677:233–243

8. Dunbar GL, Sandstrom MI, Rossignol J, Lescaudron L (2006) Neurotrophic enhancers as therapy for behavioral de fi cits in rodent models of Huntington’s disease: use of gangli-osides, substituted pyrimidines, and mesenchy-mal stem cells. Behav Cogn Neurosci Rev 5:63–79

9. Dey ND, Bombard MC, Roland BP, Davidson S, Lu M, Rossignol J, Sandstrom MI, Skeel RL, Lescaudron L, Dunbar GL (2010) Genetically engineered mesenchymal stem cells reduce behavioral de fi cits in the YAC 128 mouse model of Huntington’s disease. Behav Brain Res 214:193–200

10. Rossignol J, Boyer C, Lévèque X, Fink KD, Thinard R, Blanchard F, Dunbar GL, Lescaudron L (2011) Mesenchymal stem cell transplantation and DMEM administration in a 3NP rat model of Huntington’s disease: mor-phological and behavioral outcomes. Behav Brain Res 217:369–378

11. Rossignol J, Boyer C, Thinard R, Remy S, Dugast AS, Dubayle D, Dey ND, Boeffard F, Delecrin J, Heymann D, Vanhove B, Anegon I, Naveilhan P, Dunbar GL, Lescaudron L (2009) Mesenchymal stem cells induce a weak immune response in the rat striatum after allo

or xenotransplantation. J Cell Mol Med 13:2547–2558

12. Michel DC, Nerrière-Daguin V, Josien R, Brachet P, Naveilhan P, Neveu I (2006) Dendritic cell recruitment following xenograft-ing of pig fetal mesencephalic cells into the rat brain. Exp Neurol 202:76–84

13. von Hörsten S, Schmitt I, Nguyen HP, Holzmann C, Schmidt T, Walther T, Bader M, Pabst R, Kobbe P, Krotova J, Stiller D, Kask A, Vaarmann A, Rathke-Hartlieb S, Schulz JB, Grasshoff U, Bauer I, Vieira-Saecker AM, Paul M, Jones L, Lindenberg KS, Landwehrmeyer B, Bauer A, Li XJ, Riess O (2003) Transgenic rat model of Huntington’s disease. Hum Mol Genet 12:617–624

14. Lelan F, Boyer C, Thinard R, Rémy S, Usal C, Tesson L, Anegon I, Neveu I, Damier P, Naveilhan P, Lescaudron L (2011) Effects of human alpha-synuclein A53T-A30P mutations on SVZ and local olfactory bulb cell prolifera-tion in a transgenic rat model of Parkinson dis-ease. Parkinsons Dis 2011:987084

15. Danielyan L, Schäfer R, von Ameln-Mayerhofer A, Buadze M, Geisler J, Klopfer T, Burkhardt U, Proksch B, Verleysdonk S, Ayturan M, Buniatian GH, Gleiter CH, Frey WH 2nd (2009) Intranasal delivery of cells to the brain. Eur J Cell Biol 88:315–324

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