* These authors contributed equally
We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.
Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.
The mammalian brain has limited regenerative capacities following injury or disease, requiring innovative strategies to promote tissue and functional repair. Preclinical strategies focus on different aspects of brain regeneration, including neuroprotection, neurogenesis, angiogenesis1,2, blood-brain-barrier repair3,4, or cell therapy5,6. Cell therapy has the advantage of being able to promote many of these pro-repair processes simultaneously. In experiments with transplantation of cells, tissue repair has occurred through (1) direct cell replacement and (2) production of cytokines leading to angiogenesis and neurogenesis7. Recent advancements in stem cell technology have further facilitated the development of scalable, well-characterized neural cell sources that are now in the pipeline for clinical trials (reviewed in 7,8,9). Although cell therapies have reached the clinical stage for a few neurological diseases (e.g., Parkinson's disease10, stroke11, and spinal cord injury12), their efficacy has been variable, and more preclinical research is needed to understand the mechanisms of graft-host interactions.
One major limitation of many preclinical studies is the continuous tracking of the transplanted cells inside the host. Often only end-point measurements are performed, omitting the dynamic migratory and survival processes in the host6,13. These limitations result in the poor characterization of the grafted cells and require high animal numbers to comprehend longitudinal changes. To overcome these limitations, in this study, we transduce induced pluripotent stem cell (iPSC)-derived neural progenitor cells with a commercially available dual-reporter lentiviral vector consisting of red firefly luciferase and enhanced green fluorescent protein (rFluc-eGFP). These cells are transplanted via stereotaxic intraparenchymal injection into the mouse brain and are longitudinally tracked using in vivo bioluminescence imaging over 5 weeks. After brain tissue collection, the GFP-expressing grafted cells are identified and further characterized in histological brain sections. This method can be smoothly adapted to alternative transducable cell sources and routes of transplantation for in vivo applications in the rodent brain. Overall, the procedure is valuable to obtain longitudinal information of graft survival and migration in the mouse brain and facilitates subsequent histological characterization.
NOTE: All experiments involving mice were conducted in accordance with governmental, institutional, and ARRIVE guidelines and were approved by the Cantonal Veterinary Office of Zurich. Adult male and female non-obese diabetic SCID gamma (NSG) mice (10-14 weeks, 25-35 g) were used. Mice were housed in regular Type II/III cages in groups of at least two animals per cage in a humidity- and temperature-controlled room with a constant 12/12 h light/dark cycle. .).
1. Cell culture and viral transduction
2. Cell preparation for transplantation
3. Transplantation procedure
4. In vivo imaging
5. Perfusion
6. Processing
We aim to longitudinally track transplanted neural progenitor cells in the mouse brain using in vivo bioluminescence imaging and identify the transplanted cells in subsequent histological analysis (Figure 1A). Therefore, neural progenitor cells are transduced with a lentiviral vector consisting of EF1α-rFluc-eGFP. Before transplantation, cells were tested for successful transduction by expression of eGFP in vitro (Figure 1B). The successfully transduced cells were stereotactically transplanted in the mouse brain at the desired coordinates (e.g., in the sensorimotor cortex). Following transplantation, the mice were systemically injected with D-luciferin, the substrate for rFluc, and signal intensities of the transplanted cells were measured to confirm successful transplantation (Figure 1C).
To evaluate the detection limit of the in vivo bioluminescence imaging, a range of 6,000-180,000 cells was transplanted in the right sensorimotor cortex of the mouse (Figure 2A). We detected <6,000 cells and a bioluminescence signal proportional to the transplanted cell count directly after transplantation (Figure 2B). As human cell sources are immunogenic to immunocompetent mice, NOD scid gamma (NSG) immunodeficient mice were used to observe the long-term survival of the cell grafts. Long-term survival and detection of a bioluminescence signal for up to 5 weeks were confirmed after cell transplantation (Figure 2C,D). The transplanted cells were successfully detected ex vivo in a subsequent histological analysis through the eGFP reporter and immunostaining with anti-human nuclei and anti-human mitochondrial antibodies (Figure 2E).
Figure 1: Transplantation of neural progenitor cells. (A) Schematic overview of generation and transplantation of rFluc-eGFP NPCs. (B) Representative immunofluorescence image of transduced NPCs (GFP reporter, green) counterstained with DAPI (blue); scale bars = 5 µm. (C) In vivo detection of bioluminescence signal in transplanted cells; color bar = blue (0, min, no signal), red (4 flux, p/s × 105, max signal) Abbreviations: NPCs = neural progenitor cells; GFP = green fluorescent protein; rFluc-eGFP = red firefly luciferase and enhanced green fluorescent protein; DAPI = 4',6-diamidino-2-phenylindole; p/s = photons/s. Please click here to view a larger version of this figure.
Figure 2: Time course of transplanted cells. (A) Schematic view of cell numbers for transplantation. (B) Detection limit of transplanted cells 1 h after transplantation. (C, D) Time course of transplantation (180,000 cells) for up to 35 days in NSG mice ; color bar = blue (0, min, no signal), red (4 flux, p/s × 105, max signal) Data are mean ± SEM (n = 3). (E) Representative fluorescence images of histological sections and transplanted cells 5 weeks following transplantation. Scale bar = 10 µm. Abbreviations: D = day after transplantation; NSG = immunodeficient NOD scid gamma; DAPI = 4',6-diamidino-2-phenylindole; GFP = green fluorescent protein; HuNu = Anti-Human Nuclei Antibody, clone 235-1; p/s = photons/s. Please click here to view a larger version of this figure.
Regenerating the injured brain to allow for functional recovery remains an unmet challenge. Many innovative preclinical approaches have evolved targeting, for example, immune modulation19,20, angiogenesis1,21,22,23, blood-brain-barrier integrity2,3,24,25, and cell replacement5,26. Especially in recent years, cell-based therapies have emerged as a promising treatment strategy for the brain due to major advancements in stem cell technology and efficient differentiation protocols15,28. This paper provides a valuable protocol for transplanting and tracking neural cells in the mouse brain. The method is applicable for all transducable cell lines for in vivo applications in the mouse brain.
The presented setup uses transplants of human origin in a mouse. These transplants are not viable in the long-term in immunocompetent wild-type mice due to immunogenicity. Hence, immunodeficient NSG mice were used to overcome this limitation. Alternatively, the use of mouse transplants may be preferred to overcome the immunogenic aspects. If transplantation of human cells is required, humanized mouse models represent an emerging alternative to reduce the probability of graft rejection29.
A commercial dual-reporter viral vector consisting of firefly luciferase and eGFP under the EF1α promotor was used to visualize the transplants. This promotor was selected to achieve a high signal intensity15. However, apart from NPCs, other cell types have been shown to promote brain function after injury, including pericytes30 and astrocytes31; hence, depending on the cell line used, other promotors might be more suitable to achieve high expression levels. Additionally, the use of transgene promoters, such as CMV, may lead to downregulation, especially in long-term experiments32. The transduction efficiency of the lentiviral vector strongly depends on the used cell line and may vary between single experiments. Therefore, transduction efficiency must be evaluated before starting the in vivo experiments and to correct variations in transduction efficacy between experiments. The brain region of transplantation also influences the signal strength. Although a detection limit of <6,000 cells was achieved for cortical transplantations, it may require more cells to detect a signal in deeper brain regions, for example, striatum or hippocampus.
Transplantation volumes in the mouse brain are limited to 1-2 µL. Therefore, it is important to identify a suitable cell number for the experiments. It has been previously observed that increasing cell numbers leads to decreased survival rate, most likely due to limited availability of nutrients and oxygen in the region of transplantation33. In vivo bioluminescence imaging provides a relatively low spatial resolution compared to other in vivo imaging methods such as MRI or CT. Therefore, short migratory paths of grafted cells can only reliably be assessed in the subsequent post-hoc analysis.
The absolute signal strength of the bioluminescence is generally proportional to the transplanted cell number. However, the signal strength might be reduced if grafts are transplanted in deeper brain structures or if the signal strength is outside the linear detection spectrum of the in vivo imaging system. Currently, novel substrates are developed to ensure more efficient penetration across the blood-brain barrier than D-luciferin, including cycluc1. These substrates may further improve the detection limit of the grafted cells in the future18. Overall, this protocol allows a straightforward, minimally invasive procedure to transplant and observe grafts in the mouse brain.
The authors RR and CT acknowledge support from the Mäxi Foundation and the 3R Competence Center.
Name | Company | Catalog Number | Comments |
Viral Transduction | |||
pLL-EF1a-rFLuc-T2A-GFP-mPGK-Puro (Lenti-Labeler virus) | Systembio | LL410VA-1 | |
Consumables | |||
Eppendorf microtubes; 1.5 mL | Sigma Aldrich | Z606340 | |
Falcon Tubes; 15 mL | TPP | 91015 | |
Microscope cover slips | Product of choice | ||
Microscope slides | Product of choice | ||
Sterlie cotton swabs | Product of choice | ||
Sutures; 5/0 silk with curved needle | B. Braun | G0762482 | |
Syringe filter; 0.22 µm | TPP | 99722 | |
Syringe; 1 mL and 0.5 mL | B. Braun | 9166017V | |
Tissue culture plate (24-well) | TPP | 92024 | |
Equipment | |||
Automated cell counter (Vi-CELL XR) | Beckmann Coulter Life Science | 383721 | |
Forceps | Fine Science Tools | 11064-07 | |
Forceps, fine | Fine Science Tools | 11412-11 | |
Heating pad | Product of choice | ||
High Speed Brushless Micromotor Kit | Foredom | K.1060-22. | |
Ideal Micro Drill Burr Set Of 5 | Cell Point Specific | 60-1000 | |
In-Vivo imaging system (IVIS Lumina III with Living Imaging 4.2 software package) | Perkin Elmer | CLS136334 | |
Isoflurane vaporizer | Provet AG | 330724 | |
Microinjection Syringe Pump system | World Precision Instruments | UMP3T-1 | |
Microliter syringe; 700-Series; Volume: 5-10 µL | Hamilton | 7635-01 | |
Microtome | Leica | HM430 | |
NanoFill-33 G-Needle (removable and reusable) | World Precision Instruments | NF33BV-2 | |
Needle Holder | Fine Science Tools | 12001-13 | |
Perfusion pump and tubing | Masterflex | HV-77120-42 | |
Scalpel | Fine Science Tools | 10003-12 | |
Small bonn scissors, straight | Fine Science Tools | 14184-09 | |
Small spring scissors, straight | Fine Science Tools | 15000-03 | |
Spatula | Merck | Z243213-2EA | |
Stereotaxic frame for rodents; motorized | World Precision Instruments | 99401 | |
Pharmaceuticals and Reagents | |||
Accutase | Invitrogen | A11105-01 | Proteolytic and collagenolytic; cell dissociation reagent |
Anti-Human Nuclei Antibody, clone 235-1, Biotin Conjugate | Merck | MAB1281B | |
B27 – Supplement (50x) | Gibco | 17504-001 | |
Betadine (11 mg Iod als Povidon-Iod pro 1 ml Lösung) | Mundipharma Medical Company | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Blocking solution (3% donkey serum; 0.1% Triton-X-100 in PBS) | Product of choice; can be homemade | ||
CHIR99021 (10 mM – 2,500x) | StemMACS | 130-103-926 | |
Cryoprotectant solution | Product of choice; can be homemade | ||
DAPI solution (1 mg/mL) | Thermo Fisher Scientific | 62248 | |
D-Luciferin Potassium Salt | Perkin Elmer | 122799 | |
DMEM/F12 | Gibco | 11320-074 | |
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 | Invitrogen | A-31570 | |
Donkey serum | Product of choice | ||
Esconarcon (Pentobarbitalum natricum 300 mg) | Streuli Pharma AG | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Ethanol; 70% | Product of choice | ||
FGF Basic recombinant human protein, Animal-origin free | Thermo Fisher Scientific | PHG6015 | |
Glutamax (100x) | Gibco | A12860-01 | |
hLif (10 µg/mL – 1,000x) | PeproTech | AF-300-05-25UG | |
Isoflurane (Isofluran (1-Chlor-2,2,2-trifluorethyl-difluoromethylether) 99.9%) | Provet AG | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Laminin-L521 (L-521) | Biolaminin LN | LN521 | |
Lidocaine ointment (Lidocain: 25 mg , Prilocain: 25 mg) | Aspen Pharma Schweiz GmbH | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Mounting Medium | Product of choice; can be homemade | ||
N2- Supplement (100x) | Gibco | 75202-001 | |
Neurobasal | Gibco | 21103-049 | |
Ophtalmic lubricant (Retinol palmitat: 15,000 UI) | Bausch & Lomb Swiss AG | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Paraformaldehyde solution | Product of choice | ||
PBS | Thermo Fisher Scientific | 10010023 | Can also be homemade |
Poly-L-ornithine Solution (pLO) | Sigma-Aldrich | P4957 | |
Rimadyl (Carprofen 50 mg) | Zoetis Schweiz GmbH | All pharmaceuticals were provided by the cantonal pharmacy, Zurich, Switzerland | |
Ringer lactate | B. Braun | 3570500 | |
Ringer solution | B. Braun | 3570030 | |
Saline (0.9% NaCl) | B. Braun | 3570160 | |
SB431542 (10 mM – 3,333.3x) | StemMACS | 130-106-543 | |
Tissue Adhesive (Histoacryl) | B. Braun | 1050060 |
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