Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

Summary

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

Abstract

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)¹. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.

BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation^2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)^{8, 9}. MRI on the other hand is a non-invasive technique which is clinically applicable¹⁰ and can be used to precisely locate cellular grafts with very high resolution^11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.

In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

Protocol

1. Cell Preparation

1. Experiments should be initiated using ex vivo cultured stem cell populations genetically engineered to express the Luciferase and eGFP reporter proteins. Here we use Luciferase/eGFP-expressing murine bone marrow-derived stromal cells (BMSC-Luc/eGFP) as previously described by Bergwerf et al.^{2, 5}.
2. Two days before cell labeling, plate BMSC-Luc/eGFP cells at a density of 8 x 10⁵ cells per T75 culture flask in 15 ml complete expansion medium (CEM) supplemented with 1 μg/ml Puromycine.
3. Allow cells to grow for 48 hr at 37 °C and 5% CO₂.
4. Add 5 x 10⁶ Glacial Blue....

Discussion

In this report, we describe an optimized protocol for the combination of three complementary imaging modalities (BLI, MRI and histology) for detailed characterization of cellular implants in the CNS of immune competent mice. A combination of reporter gene labeling of cells, based on genetic modification with the reporter genes firefly Luciferase and eGFP, and a direct cell labeling with GB MPIO, leads to an accurate assessment of stem cell grafts in vivo.

For particle labeling of BMSC.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IMDM	Lonza Inc.	BE12-722F	Component of the cell growth medium CEM
Fetal bovine serum	GIBCO, by Life Technologies	10270-106	Component of the cell growth medium CEM
Horse serum	GIBCO, by Life Technologies	1605-122	Component of the cell growth medium CEM
Penicillin-streptomycin	GIBCO, by Life Technologies	15140	Component of the cell growth medium CEM
Fungizone	GIBCO, by Life Technologies	15290-018	Component of the cell growth medium CEM
PBS	GIBCO, by Life Technologies	14190
Puromycine	Invitrogen	ant-pr-1
trypsin	GIBCO, by Life Technologies	25300
GB MPIO	Bangs Laboratories	ME04F/7833
D-luciferin	Promega Corp.	E1601
Ketamine (Ketalar)	Pfizer Pharma GmbH
Xylazine (Rompun)	Bayer AG
Isoflurane	Isoflo	05260-05
0.9% NaCl solution	Baxter Internationl Inc.
paraformaldehyde	Merck & Co., Inc.	1.04005.1000
sucrose	Applichem	A1125
Micro-injection pump	KD scientific	KDS100
Photon imager	Biospace Lab
9.4T MR scanner	Bruker Corporation	Biospec 94/20 USR
BX51 microscope	Olympus Corporation	BX51
Mycrom HM cryostat	Prosan	HM525
syringe	Hamilton Co	7635-01
30 gauge needle	Hamilton Co	7762-03
Photo Vision software	Biospace Lab
M3vision software	Biospace Lab
Paravision 5.1 software	Bruker Corporation
Amira 4.0 software	Visage Imaging