multimodal-imaging-stem-cell-implantation-central-nervous-system

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

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice.

SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.

The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells.

While neural stem cells (NSCs) are widely expected to become a therapeutic agent for treatment of severe injuries to the central nervous system (CNS), currently there are only few detailed preclinical studies linking cell fate with experimental outcome. In this study, we aimed to validate whether IV administration of allogeneic NSC can improve experimental autoimmune encephalomyelitis (EAE), a well-established animal model for human multiple sclerosis (MS). For this, we cultured adherently growing luciferase-expressing NSCs (NSC-Luc), which displayed a uniform morphology and expression profile of membrane and intracellular markers, and which displayed an in vitro differentiation potential into neurons and astrocytes. Following labeling with green fluorescent micron-sized iron oxide particles (f-MPIO-labeled NSC-Luc) or lentiviral transduction with the enhanced green fluorescent protein (eGFP) reporter gene (NSC-Luc/eGFP), cell implantation experiments demonstrated the intrinsic survival capacity of adherently cultured NSC in the CNS of syngeneic mice, as analyzed by real-time bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histological analysis. Next, EAE was induced in C57BL/6 mice followed by IV administration of NSC-Luc/eGFP at day 7 postinduction with or without daily immunosuppressive therapy (cyclosporine A, CsA). During a follow-up period of 20 days, the observed clinical benefit could be attributed solely to CsA treatment. In addition, histological analysis demonstrated the absence of NSC-Luc/eGFP at sites of neuroinflammation. In order to investigate the absence of therapeutic potential, BLI biodistribution analysis of IV-administered NSC-Luc/eGFP revealed cell retention in lung capillaries as soon as 1-min postinjection, resulting in massive inflammation and apoptosis in lung tissue. In summary, we conclude that IV administration of NSCs currently has limited or no therapeutic potential for neuroinflammatory disease in mice, and presumably also for human MS. However, given the fact that grafted NSCs have an intrinsic survival capacity in the CNS, their therapeutic exploitation should be further investigated, and-in contrast to several other reports-will most likely be highly complex.

Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice?

Currently, much attention is given to the development of cellular therapies for treatment of central nervous system (CNS) injuries. Diverse cell implantation strategies, either to directly replace damaged neural tissue or to create a neuroregenerative environment, are proposed to restore impaired brain function. However, because of the complexity of the CNS, it is now becoming clear that the contribution of cell implantation into the brain will mainly act in a supportive manner. In addition, given the time dependence of neural development during embryonic and post-natal life, cellular implants, either self or non-self, will most likely have to interact for a sustained period of time with both healthy and injured neural tissue. The latter also implies potential recognition of cellular implants by the innate immune system of the brain. In this review, we will emphasize on preclinical observations in rodents, regarding the recognition and immunogenicity of autologous, allogeneic and xenogeneic cellular implants in the CNS of immune-competent hosts. Taken together, we here suggest that a profound study of the interaction between cellular grafts and the brain's innate immune system will be inevitable before clinical cell transplantation in the CNS can be performed successfully.

Recognition of cellular implants by the brain's innate immune system.

Development of multimodal imaging strategies is currently of utmost importance for the validation of preclinical stem cell therapy studies.

Labeling of Luciferase/eGFP-expressing bone marrow-derived stromal cells with fluorescent micron-sized iron oxide particles improves quantitative and qualitative multimodal imaging of cellular grafts in vivo.

Transplantation of neural stem cells (NSC) is hoped to become a promising primary or secondary therapy for the treatment of various neurodegenerative disorders of the central nervous system (CNS), as demonstrated by multiple pre-clinical animal studies in which functional recovery has already been demonstrated. However, for NSC therapy to be successful, the first challenge will be to define a transplantable cell population. In the first part of this review, we will briefly discuss the main features of ex vivo culture and characterisation of NSC. Next, NSC grafting itself may not only result in the regeneration of lost tissue, but more importantly has the potential to improve functional outcome through many bystander mechanisms. In the second part of this review, we will briefly discuss several pre-clinical studies that contributed to a better understanding of the therapeutic potential of NSC grafts in vivo. However, while many pre-clinical animal studies mainly report on the clinical benefit of NSC grafting, little is known about the actual in vivo fate of grafted NSC. Therefore, the third part of this review will focus on non-invasive imaging techniques for monitoring cellular grafts in the brain under in vivo conditions. Finally, as NSC transplantation research has evolved during the past decade, it has become clear that the host micro-environment itself, either in healthy or injured condition, is an important player in defining success of NSC grafting. The final part of this review will focus on the host environmental influence on survival, migration and differentiation of grafted NSC.

Current challenges for the advancement of neural stem cell biology and transplantation research.

Stem cell transplantation holds great promise for restoration of neural function in various neurodegenerative disorders, including multiple sclerosis (MS). However, many questions remain regarding the true efficacy and precise mode of action of stem cell-based therapeutic approaches. Therefore, in this article, we will first discuss the ideal route and/or timing of stem cell-based therapies for experimental autoimmune encephalomyelitis (EAE), the most used preclinical animal model for MS. Next, we will provide an overview of the proposed mechanisms that contribute to the beneficial effects of stem cell transplantation observed during the treatment of rodent EAE. Reviews of current and past literature clearly demonstrate conceptual changes in the development of stem cell-based approaches for EAE/MS, leading to the identification of several major challenges to be tackled before (stem) cell therapy for rodent EAE can be safely and successfully translated to human therapy for MS.

Stem cell therapy for multiple sclerosis: preclinical evidence beyond all doubt?

Cell transplantation has been suggested to display several neuroprotective and/or neuroregenerative effects in animal models of central nervous system (CNS) trauma. However, while most studies report on clinical observations, currently little is known regarding the actual fate of the cell populations grafted and whether or how the brain's innate immune system, mainly directed by activated microglia and astrocytes, interacts with autologous cellular implants. In this study, we grafted well-characterized neural stem cell, mouse embryonic fibroblast, dendritic cell, bone marrow mononuclear cell, and splenocyte populations, all isolated or cultured from C57BL/6-eGFP transgenic mice, below the capsula externa (CE) of healthy C57BL/6 mice and below the inflamed/demyelinated CE of cuprizone-treated C57BL/6 mice. Two weeks postgrafting, an extensive quantitative multicolor histological analysis was performed in order (i) to quantify cell graft localization, migration, survival, and toxicity and (ii) to characterize endogenous CNS immune responses against the different cell grafts. Obtained results indicate dependence on the cell type grafted: (i) a different degree of cell graft migration, survival, and toxicity and (ii) a different organization of the endogenous immune response. Based on these observations, we warrant that further research should be undertaken to understand-and eventually control-cell graft-induced tissue damage and activation of the brain's innate immune system. The latter will be inevitable before cell grafting in the CNS can be performed safely and successfully in clinical settings.

Article	Year
BMC biotechnology\| PubMed ID: 18078525	2007
Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice. BMC biotechnology\| PubMed ID: 19128466	2009
Immunology and cell biology\| PubMed ID: 19290016	2009
The ratio of SRPK1/SRPK1a regulates erythroid differentiation in K562 leukaemic cells. Biochimica et biophysica acta\| PubMed ID: 20708644	2010
Clinical potential of intravenous neural stem cell delivery for treatment of neuroinflammatory disease in mice? Cell transplantation\| PubMed ID: 21092405	2011
Recognition of cellular implants by the brain's innate immune system. Immunology and cell biology\| PubMed ID: 21102538	2011
Labeling of Luciferase/eGFP-expressing bone marrow-derived stromal cells with fluorescent micron-sized iron oxide particles improves quantitative and qualitative multimodal imaging of cellul... Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging\| PubMed ID: 21246293	2011
Current challenges for the advancement of neural stem cell biology and transplantation research. Stem cell reviews\| PubMed ID: 21537994	2012
Stem cell therapy for multiple sclerosis: preclinical evidence beyond all doubt? Regenerative medicine\| PubMed ID: 22397612	2012
Cell type-associated differences in migration, survival, and immunogenicity following grafting in CNS tissue. Cell transplantation\| PubMed ID: 22472278	2012

Jasmijn Daans

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