The authors thank Jayden Smith for critically reviewing and proof editing the manuscript. This work has received support from the National Multiple Sclerosis Society (NMSS, partial grants RG-4001-A1), the Italian Multiple Sclerosis Association (AISM, grant 2010/R/31), the Italian Ministry of Health (GR08-7), Wings for Life, Banca Agricola Popolare di Ragusa (BAPR), the European Research Council (ERC) under the ERC- 2010-StG Grant agreement no&#160;260511-SEM_SEM and the European Community (EC) 7th Framework Program (FP7/2007-2013) under Grant Agreement n*deg; 280772- iONE.

<ol>
	<li>Ben-Hur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplanted+multipotential+neural+precursor+cells+migrate+into+the+inflamed+white+matter+in+response+to+experimental+autoimmune+encephalomyelitis.">Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis.</a> Glia. 41, 73-80 (2003).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+adult+neurospheres+induces+recovery+in+a+chronic+model+of+multiple+sclerosis.">Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis.</a> Nature. 422, 688-694 (2003).</li><li>Chu, K., Kim, M., Jeong, S. W., Kim, S. U., Yoon, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+can+migrate,+differentiate,+and+integrate+after+intravenous+transplantation+in+adult+rats+with+transient+forebrain+ischemia.">Human neural stem cells can migrate, differentiate, and integrate after intravenous transplantation in adult rats with transient forebrain ischemia.</a> Neurosci. Lett. 343, 129-133 (2003).</li><li>Bottai, D., Madaschi, L., Di Giulio, A. M., Gorio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability-dependent+promoting+action+of+adult+neural+precursors+in+spinal+cord+injury.">Viability-dependent promoting action of adult neural precursors in spinal cord injury.</a> Mol. Med. 14, 634-644 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurosphere-derived+multipotent+precursors+promote+neuroprotection+by+an+immunomodulatory+mechanism.">Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism.</a> Nature. 436, 266-271 (2005).</li><li>Einstein, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraventricular+transplantation+of+neural+precursor+cell+spheres+attenuates+acute+experimental+allergic+encephalomyelitis.">Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis.</a> Mol. Cell Neurosci. 24, 1074-1082 (2003).</li><li>Chu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cell+transplantation+reduces+spontaneous+recurrent+seizures+following+pilocarpine-induced+status+epilepticus+in+adult+rats.">Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats.</a> Brain Res. 1023, 213-221 (2004).</li><li>Jeong, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cell+transplantation+promotes+functional+recovery+in+rats+with+experimental+intracerebral+hemorrhage.">Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage.</a> Stroke. 34, 2258-2263 (2003).</li><li>Aboody, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+display+extensive+tropism+for+pathology+in+adult+brain:+evidence+from+intracranial+gliomas.">Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12846-12851 (2000).</li><li>Muller, F. J., Snyder, E. Y., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy:+can+neural+stem+cells+deliver.">Gene therapy: can neural stem cells deliver.</a> Nat. Rev. Neurosci. 7, 75-84 (2006).</li><li>Martino, G., Pluchino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+therapeutic+potential+of+neural+stem+cells.">The therapeutic potential of neural stem cells.</a> Nat. Rev. Neurosci. 7, 395-406 (2006).</li><li>Deckert-Schluter, M., Schluter, D., Hof, H., Wiestler, O. D., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+ICAM-1,+VCAM-1+and+their+ligands+LFA-1,+Mac-1,+CD43,+VLA-4,+and+MHC+class+II+antigens+in+murine+Toxoplasma+encephalitis:+a+light+microscopic+and+ultrastructural+immunohistochemical+study.">Differential expression of ICAM-1, VCAM-1 and their ligands LFA-1, Mac-1, CD43, VLA-4, and MHC class II antigens in murine Toxoplasma encephalitis: a light microscopic and ultrastructural immunohistochemical study.</a> J. Neuropathol. Exp. Neurol. 53, 457-468 (1994).</li><li>Hemmer, B., Archelos, J. J., Hartung, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+the+immunopathogenesis+of+multiple+sclerosis.">New concepts in the immunopathogenesis of multiple sclerosis.</a> Nat. Rev. Neurosci. 3, 291-301 (2002).</li><li>Butcher, E. C., Picker, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+homing+and+homeostasis.">Lymphocyte homing and homeostasis.</a> Science. 272, 60-66 (1996).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+systemic+delivery+of+neural+precursor+cells+to+the+brain:+assembly+of+brain+endothelial+apical+cups+and+control+of+transmigration+by+CD44.">Molecular mechanism of systemic delivery of neural precursor cells to the brain: assembly of brain endothelial apical cups and control of transmigration by CD44.</a> Stem Cells. 26, 1673-1682 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+ameliorate+autoimmune+encephalomyelitis+in+non-human+primates.">Human neural stem cells ameliorate autoimmune encephalomyelitis in non-human primates.</a> Ann. Neurol. 66, 343-354 (2009).</li><li>Martino, G., Pluchino, S., Bonfanti, L., Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+regeneration+in+physiology+and+pathology:+the+immune+signature+driving+therapeutic+plasticity+of+neural+stem+cells.">Brain regeneration in physiology and pathology: the immune signature driving therapeutic plasticity of neural stem cells.</a> Physiol. Rev. 91, 1281-1304 (2011).</li><li>Aharonowiz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+transplanted+human+embryonic+stem+cell-derived+neural+precursors+in+an+animal+model+of+multiple+sclerosis.">Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis.</a> PLoS ONE. 3, e3145 (2008).</li><li>Pluchino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulatory+neural+stem/precursor+cells+protect+from+central+nervous+system+autoimmunity+by+restraining+dendritic+cell+function.">Immune regulatory neural stem/precursor cells protect from central nervous system autoimmunity by restraining dendritic cell function.</a> PLoS One. 4, (2009).</li><li>Einstein, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+precursors+attenuate+autoimmune+encephalomyelitis+by+peripheral+immunosuppression.">Neural precursors attenuate autoimmune encephalomyelitis by peripheral immunosuppression.</a> Ann. Neurol. 61, 209-218 (2007).</li><li>Gritti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotential+stem+cells+from+the+adult+mouse+brain+proliferate+and+self-renew+in+response+to+basic+fibroblast+growth+factor.">Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.</a> J. Neurosci. 16, 1091-1100 (1996).</li><li>Furlan, R., Pluchino, S., Marconi, P. C., Martino, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+gene+delivery+into+the+central+nervous+system+using+intrathecally+injected+nonreplicative+viral+vectors.">Cytokine gene delivery into the central nervous system using intrathecally injected nonreplicative viral vectors.</a> Methods Mol. Biol. 215, 279-289 (2003).</li><li>Constantin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+and+analysis+of+adhesive+events+in+brain+microvessels+by+using+intravital+microscopy.">Visualization and analysis of adhesive events in brain microvessels by using intravital microscopy.</a> Methods Mol. Biol. 239, 189-198 (2004).</li><li>Politi, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic-resonance-based+tracking+and+quantification+of+intravenously+injected+neural+stem+cell+accumulation+in+the+brains+of+mice+with+experimental+multiple+sclerosis.">Magnetic-resonance-based tracking and quantification of intravenously injected neural stem cell accumulation in the brains of mice with experimental multiple sclerosis.</a> Stem Cells. 25, 2583-2592 (2007).</li><li>Melzi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-graft+of+allogeneic+immune+regulatory+neural+stem+cells+(NPC)+and+pancreatic+islets+mediates+tolerance,+while+inducing+NPC-derived+tumors+in+mice.">Co-graft of allogeneic immune regulatory neural stem cells (NPC) and pancreatic islets mediates tolerance, while inducing NPC-derived tumors in mice.</a> PLoS One. 5, (2010).</li><li>Amariglio, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-derived+brain+tumor+following+neural+stem+cell+transplantation+in+an+ataxia+telangiectasia+patient.">Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.</a> PLoS Med. 6, (2009).</li><li>Ben-Hur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+proinflammatory+cytokines+on+the+growth,+fate,+and+motility+of+multipotential+neural+precursor+cells.">Effects of proinflammatory cytokines on the growth, fate, and motility of multipotential neural precursor cells.</a> Mol. Cell Neurosci. 24, 623-631 (2003).</li><li>Giusto, E., Donega, M., Cossetti, C., Pluchino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-immune+interactions+of+neural+stem+cell+transplants:+From+animal+disease+models+to+human+trials.">Neuro-immune interactions of neural stem cell transplants: From animal disease models to human trials.</a> Exp. Neurol. , (2013).</li></ol>

The authors have nothing to disclose.

Somatic stem cell based therapies are emerging as one of the most promising strategies for treating chronic inflammatory CNS disorders such as MS211. While the mechanisms sustaining their therapeutic effects still need to be completely elucidated, the significant impact of NPC transplantation in different experimental models of neurodegenerative diseases has given rise to the somewhat provocative belief that stem cells may soon be applied into human studies. However, before envisaging any potential human applications of such innovative therapies we need to face some key issues and address some unanswered questions, such as the identification of the ideal stem cell source for transplantation (autologous vs allogeneic, pluripotent vs multipotent) and the best route of administration.
Neurodegenerative diseases differ in their pathophysiologies, with some being spatially confined (e.g. spinal cord injury, stroke) while other being characterized by a spatial temporal dissemination (e.g. MS). It derives that the focal injection of (stem) cells may be a suitable treatment for the former, while it may be inadequate or simply unfeasible for the latter, where the presence of multiple sites of brain damage represents an additional challenge to be overcome.
Consistent evidence has shown that intravenous delivery may represent a valid (and low invasive) protocol for cell administration, supported by the intrinsic ability of stem cells to sense the environment and specifically home towards the site(s) of damage. However, translation of systemic NPC therapies into clinics is still hindered by a few limitations, such as the possible accumulation of transplanted cells in peripheral non-CNS organs (e.g. lungs, spleen, lymph nodes, and kidneys) that may or may not be sites of local inflammatory responses in vivo. While this nonspecific accumulation of injected NPCs out of the target CNS is quickly cleared in mice with chronic EAE2, there is evidence of long term NPC persistence (or in some cases exclusive targeting) at the level of the secondary lymphoid organs (e.g. lymph&#160;nodes and spleen) after systemic injection in mice with relapsing EAE519,20. The fact that significant numbers of transplanted NPCs recirculate toward non-CNS organs creates a dilution effect, inevitably reducing the absolute number of NPCs in the CNS. Interestingly, systemically injected NPCs are therapeutically efficacious (e.g. via immune regulatory actions) even when accumulating exclusively out of the CNS19,20. And the overall therapeutic effects of injected NPCs targeting the CNS only5&#160;or the lymph&#160;nodes only19&#160;are comparable. This may in part due to an intrinsic plasticity of the cells, which respond differently to the molecular components of the surrounding microenvironment. Notably, if on one hand transplanted NPCs may feel the presence of inflammatory cytokines and exert their therapeutic effect through immune modulatory mechanisms16, on the other hand they may come in contact with hostile microenvironments ultimately leading to the formation of tumors25,26. For these reasons, the preferred option is at present to concentrate injected NPCs in the CNS. As such, the intrathecal administration (via a lumbar or cisternal route) is still the most accepted prospective route of administration in clinical trials. However, the multifocality and pathological heterogeneity of MS lesions may limit the efficacy of such an approach.
Here we describe an efficient protocol to isolate, maintain and characterize mouse neural progenitors from the adult SVZ and successively, how to systemically (both i.v and i.c.v) inject NPCs in mice affected by chronic EAE, one of the most studied models of MS. Even though relatively simple and straightforward, these protocols present some critical steps that need to be taken into consideration. At first, it is mandatory to obtain a stably expandable and healthy cell preparation. As described throughout the protocol, the stability of the cells may be indirectly inferred by looking at their growth rate. Ideally,&#160;neurospheres should reach a diameter of 150-200 &#956;m every 4-5 DIV and the percentage of cell death upon passages must be very low (less than 10%). Also, neurospheres must present a compact and regular round shape at the microscope. The infection with lentiviruses may interfere with the survival and stability of the cells. To obtain an optimal infection, it is mandatory to obtain a titration of the virus of interest sufficient to obtain 3 x 106 T.U./ml. Indeed, while on one hand a low amount of virus would lead to a small percentage of infected cells, on the other hand the use of a high amount would likely lead to a toxicity effect (strongly dependent on the gene to be infected). NPCs can be infected multiple times with integrating viruses, should the outcome of the first transduction lead to low percentage of transgene positive viable cells. It is always a good practice to have intra experimental comparison of stability and viability parameters with wild type, nontransduced, controls NPCs. As for the induction of chronic EAE, the most problematic part is represented by the preparation of the emulsion. As described, glassware and ice must be use at all times. The preparation of the emulsion takes 30-45 min, and it can be considered ready to be injected only when it does not disperse when dropped on water. If the emulsion dissipates in water, this means it is not yet ready and requires further blending.
Good accuracy during the i.v. injection procedure at the time of cell administration is critical. First, it is extremely important to have a single cell suspension, as the injection of aggregated cells may obstruct the veins of the injected mouse and lead to its immediate death. To assure the correct positioning of the needle into the vein, it is good practice to aspirate with the syringe few microliters of blood. In the case of no blood coming into the syringe, the operator should slowly move the needle backward or forward until the correct location is found. Only at this point can the cell suspension be slowly injected. Importantly, the operator should typically not be required to apply any pressure on the syringe since the liquid should easily flow in the vein. An improper injection would inevitably result in a subcutaneous swelling corresponding to the injection site.
It is worth noting that in the very near future systemically injected NPCs2,27, genetically modified or not, may themselves represent a therapeutic option, as well as providing an important tool for delivering immune modulatory and/or neuroprotective drugs and pro remyelinating agents directly into the CNS28. Therefore, while some limitations related to the systemic delivery of NPCs do exist, both i.v. and i.c.v. routes may ultimately represent valid alternative therapeutic approach in those diseases where the focal injection of cells may not constitute the gold standard protocol of treatment.

Strong evidence has arisen from in vivo studies attesting to the therapeutic efficacy of the transplantation of somatic neural stem/precursor cells (NPCs) in animal models of CNS disorders1-8. Nevertheless, a number of issues relating to the delivery of stem cells into the host require careful consideration before these experimental results can be translated into clinical applications. A particularly substantial hurdle towards the development of (nonhematopoietic) restorative stem cell therapies for multifocal, chronic inflammatory brain diseases is the identification of the ideal route of NPC injection. A firm understanding of the pathophysiology of the targeted disease (focal or multifocal; primary inflammatory or primary degenerative), and a cautious analysis of feasibility and risk issues associated with the delivery techniques are in identifying the optimal protocol for stem cell delivery.
While the focal (e.g. into the nervous system parenchyma) stem cell transplantation is a logical approach to the treatment of CNS diseases characterized by spatially confined areas of damage (e.g. Parkinson's and Huntington's disease, brain and spinal cord traumatic injuries, and stroke), the very same approach may prove to be practically not feasible in conditions such as MS, where a multifocal, chronic, and spatially disseminated CNS damage accumulates over time. In this latter case, targeting focal cell injections to individual lesions is also hindered by the limited capacity of transplanted NPCs to migrate over long distances within the CNS parenchyma, thus prompting the identification of alternative, more suitable methods of CNS targeting with less invasive NPC transplants.
Great promise emerged from the observations that NPCs target an intracranial tumor (e.g. glioma) in mice when injected intravascularly outside the CNS9. Following this seminal in vivo evidence of the stem cell pathotrophism10, extensive data have been accumulated pertaining to the feasibility and therapeutic efficacy of the systemic transplantation of NPCs in laboratory animals with experimental autoimmune encephalomyelitis (EAE), as a model of inflammatory CNS damage, via either intravenous (i.v.) or intracerebroventricular (i.c.v.) NPC injection1,2,5,6,8.. We have first shown that this is dependent on the capability of transplanted NPCs to target and enter the inflamed CNS, and to subsequently engage multiple intercellular communications programs within specific microenvironments in vivo11. In order to specifically target the CNS, NPCs are delivered directly into the cerebrospinal fluid (CSF) circulation by i.c.v. injection, or into the bloodstream via i.v. injection. Once entering either the bloodstream or CSF, transplanted NPCs actively interact with the blood brain (BBB) or blood cerebrospinal fluid (BCSFB) barriers and enter the CNS parenchyma. This interaction between the NPC graft and the BBB (or BCSFB) is regulated by specific set of NPC surface cell adhesion molecules (CAMs) and facilitated by the expression of high levels of CAM counter-ligands on activated endothelial/ependymal cells12-14. Examples of these CAMs include the receptor for hyaluronate, CD44, and the intercellular adhesion molecule (ICAM)-1 ligand very late antigen (VLA)-45,15,16 (that, in leukocytes, are responsible of the interaction with activated ependymal and endothelial cells), and to a much lower extent Lymphocyte function-associated antigen (LFA)-1 and P-selectin glycoprotein ligand (PSGL)-1. NPCs also express a wide range of chemokine receptors, including CCR1, CCR2, CCR5, CXCR3, and CXCR4 (but do not express CCR3 and CCR7), which are functionally active, both in vitro and in vivo5,16. Thus, systemically injected NPCs use these CAMs, along with G-protein coupled receptor (GPCRs), to accumulate at the level of the inflamed CNS. Conversely, NPCs injected systemically into healthy mice do not enter the CNS via vascular or cerebrospinal fluid space routes2. CNS inflammation, or endothelial/ependymal cell activation following systemic cytokine or lypopolisaccharide (LPS) injection as a model of chemically induced encephalitis, is therefore necessary for the accumulation of systemically injected NPCs into the brain and spinal cord2. Thus, successful targeting of the CNS with systemic NPC therapies is dependent on the identification of a disease specific window of Opportunity (WoO) in which the brain and spinal cord environment are conducive to the accumulation and transendothelial migration of NPCs. Such conditions generally arise in the context of acute and subacute inflammation17. Once having entered the CNS, transplanted undifferentiated NPCs have been shown to ameliorate the clinico-pathological features of mice as well as larger, nonhuman primates with EAE. This has been described to be dependent from minimal cell replacement2 and remarkable secretion of immune regulatory and neuroprotective paracrine factors within perivascular CNS2,5,6,18 vs non-CNS inflamed areas19,20 (e.g. lymph nodes) in response to the inflammatory cell signaling elicited by infiltrating immune cells5.
Herein we describe the key methodological aspects of the systemic injection of somatic NPCs into a mouse model of chronic EAE. More specifically, we define the protocols that we have established to (i) derive, expand and prepare for transplantation somatic NPCs from the subventricular zone (SVZ) of adult C57BL/6 mice; (ii) induce chronic EAE in such mice and (iii) perform therapeutically efficacious systemic (i.v. or i.c.v) NPC transplantation into EAE mice.

<table border="0" cellpadding="0" cellspacing="0" width="1474">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:753px;">Cell culture</td>
			<td style="width:300px;"></td>
			<td style="width:149px;"></td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EBSS</td>
			<td>Sigma</td>
			<td>E2888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Cystein</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papain</td>
			<td>WORTHINGTON</td>
			<td>30H11965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher scientific</td>
			<td>D/0700/50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse NeuroCult basal medium</td>
			<td>Stem Cell technologies</td>
			<td>05700</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult proliferation supplements</td>
			<td>Stem Cell technologies</td>
			<td>05701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Basic fibroblast growth factor</td>
			<td>Peprotech</td>
			<td>100-18B-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epidermal growth factor</td>
			<td>Peprotech</td>
			<td>AF-100-15-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td>1514012</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel (coating solution)</td>
			<td>BD biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NeuroCult&#174; Differentiation Kit (Mouse)</td>
			<td>Stem cell technologies</td>
			<td>05704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Accumax</td>
			<td>eBioscience</td>
			<td>00-4666-56</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS (DPBS) (10x) without Ca&#38; Mg</td>
			<td>PAA LABORATORIES LTD</td>
			<td>H15-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Myco trace</td>
			<td>PAA LABORATORIES LTD</td>
			<td>Q052-020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide (DMSO)</td>
			<td>SIGMA</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>PAA LABORATORIES LTD</td>
			<td>B11-035</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Nestin</td>
			<td>Abcam</td>
			<td>ab11306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti GFAP</td>
			<td>DAKO</td>
			<td>203344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti Histone H3 (phospho S10)&#160;</td>
			<td>Abcam</td>
			<td>ab14955</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti MAP-2</td>
			<td>Abcam</td>
			<td>ab32454</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti MBP</td>
			<td>AbD SEROTEC</td>
			<td>MCA409S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-O4 Antibody, clone 81 | MAB345</td>
			<td>Millipore</td>
			<td>MAB345</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mounting solution</td>
			<td>DAKO</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EAE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freund&#39;s Adjuvant Incomplete</td>
			<td>SIGMA-ALDRICH CO LTD</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mycobacterium tuberculosis&#160;</td>
			<td>DIFCO</td>
			<td>H37Ra</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MOG(35&#8211;55)&#160;</td>
			<td>Espikem</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pertussis toxin</td>
			<td>List Biological Laboratories</td>
			<td>181</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iris scissor straight</td>
			<td>Fine Sciences Tolls</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Blunt/bended forceps</td>
			<td>Fine Sciences Tolls</td>
			<td>11080-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Brain slicer</td>
			<td>Zivic instruments</td>
			<td>BSMAS005-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blades</td>
			<td>Swann-Morton</td>
			<td>324</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P200, P1000 pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine (Vetalar)</td>
			<td style="width:300px;">Boehringer Ingelheim</td>
			<td>01LC0030&#160;&#160;</td>
			<td style="width:272px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine (Rompun)</td>
			<td>Bayer</td>
			<td>32371</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereotaxic frame</td>
			<td>KOPF</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7762-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde (PFA)</td>
			<td>SIGMA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASTAIN Elite ABC Kit</td>
			<td>vector laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
	</tbody>
</table>

systemic injection of neural stem/progenitor cells in mice with chronic eae

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

NPC derivation and characterization 
SVZ dissections are performed on pools (n= 5-7 mice/pool) of 6-8 week old C57Bl/6 mice by means of mechanical and enzymatic dissociation (Figure 1A). After a few days of culturing in CGM, free-floating neurospheres begin forming (Figures 1A and 1B). Primary spheres are collected and mechanically passaged every 4-5 DIV. Upon passaging, numbers of live and dead cells are ascertained and cumulative cell numbers plotted to generate a growth curve (Figure 1C). This gives an indication of the propagation rate, thus providing an indirect parameter to evaluate the global stability of the NPC preparation. When proliferating as neurospheres, NPCs express markers of mitotic activity (phospho-histone H3, PHH3; 5 &#956;g/ml) and of undifferentiated neural cells (e.g. Nestin) (Figure 2A). Undifferentiated NPCs that are to be therapeutically efficacious in EAE after transplantation must express cell surface adhesion molecules that include CD44 (5 &#956;g/ml), the &#945;4 subunit of the VLA-4 (10 &#956;g/ml) (Figure 2F). When plated under appropriate differentiation conditions, NPCs express markers typical of the three neural lineages, (Figure 2B), such as the astroglial marker GFAP (Figure 2C), the neuronal marker microtubule associated protein-2 (MAP-2; 5 &#956;g/ml) (Figure 2D) and the oligodendroglial markers O4 (5 &#956;g/ml) and myelin basic protein (MBP; 10 &#956;g/ml) (Figure 2E), thus confirming that NPCs are multipotent towards the three main neural lineages. To facilitate the identification of transplanted cells in vivo, NPCs are transduced with lentiviruses engineered to stably express reporter genes such as GFP. Transduced NPCs express GFP both as proliferating neurospheres (Figures 3A and 3C) as well as when differentiating in vitro upon growth factor withdrawal (Figure 3B). To verify the efficiency of transduction, the expression of GFP is analyzed by flow cytometry, starting as early as 3 passages after cell transduction (that is allowing enough time to have robust expression of the introduced transgene at the protein level). When applying third generation lentiviruses to NPCs in vitro, we consistently observe &#62;90% of cells showing high levels of GFP over multiple independent experiments (Figure 3C), with neither overt toxicity nor changes in proliferation, when comparing the growth curves of wild type and lenti-GFP transduced NPCs (Figure 3D).
Chronic EAE induction 
EAE is one of the best-characterized models of MS, since it recapitulates most of the pathological and clinical events of MS. Immunization of C57Bl/6 mice with MOG35-55 leads to the development of a chronic form of EAE. When approaching the disease onset (11.3&#177;1.8 dpi) during the induction phase (preclinical), MOG immunized mice start losing weight (Figure 4A). At the beginning of the effector/invasion phase (peak clinical; 16.8&#177;3.2 dpi) EAE mice reach the peak of the disease (score 3.5&#177;0.7), and shortly after peaking they show a progressive and partial recovery that finally stabilizes during the chronic phase (stable clinical) from around 30 dpi onwards (score 2.8&#177;0.9) (Figure 4B). Numerous studies, including some investigating EAE by either intravital microscopy23 or magnetic resonance imaging24, paired with in vivo tissue pathology, have identified in the peak of the effector/invasion phase (16-22 dpi) the ideal window of opportunity (WoO) in which to perform systemic experimental therapies with stem cells intended to enter the chronically inflamed EAE CNS and protect it from secondary damage.
NPC injection 
Syngeneic NPCs injected systemically in EAE mice (Figure 6) are found almost exclusively in perivascular areas of CNS damage, both in the brain (Figure 5A) and spinal cord (Figures 5B-D), up to 45 days post transplantation (dpt), as described5. i.v. injected NPCs preferentially retain an immature, Nestin+ (Figure 5C) phenotype, while few NPCs moving out of the perivascular area express NeuN (Figure 5D). Of note, NPCs injected i.v. into healthy controls fail to enter the CNS via the vascular route (Figure 5E). After i.v. NPC injection, significant numbers of injected NPCs are found at the level of non-CNS organs such as the liver, gut, spleen, lung and kidney, but not the heart, as early as 10 dpt (Figure 5F). Non-CNS accumulating NPCs are completely cleared out of these peripheral organs by 30 dpt (Figure 5G).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig1highres.jpg" src="/files/ftp_upload/51154/51154fig1.jpg" /> 
Figure 1. Isolation of NPCs from the SVZ of adult mice and generation of stably expandable NPC lines. A, Schematic representation of the main critical steps of the generation of stably expandable, and preparation of injectable mouse NPCs. B, Neurospheres in vitro. C, NPCs cultured as neurospheres are passaged every 4-5 days. Numbers of live and dead cells are collected and cumulative cell numbers plotted to generate a growth curve. The scale bar in B is 200 &#956;m. Data in C are mean cumulative number of cells &#177; SD (n= 3).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig2highres.jpg" src="/files/ftp_upload/51154/51154fig2.jpg" /> 
Figure 2. NPCs characterization. A, proliferating mouse neurospheres express the PHH3 core histone protein (green), and the type VI intermediate filament protein Nestin (red). B-E, After 6 DIV in differentiation conditions, NPCs are multipotent towards astroglial (GFAP, red in C), neuronal (MAP-2, green in D) and oligodendroglial (O4, green in E; and MBP, red in E) lineages. Quantification of GFAP-, MAP-2-, and O4-expressing cells at 6 DIV after NPC differentiation in vitro are showed in B. Data in B are as mean % &#177; SD (n= 3). The scale bars in C-E are 50 &#956;m. F, Flow cytometry analysis of the expression of the main cell surface adhesion molecules regulating leukocyte extravasation in mouse neurospheres. Figure 2F is reproduced from Pluchino et al.2
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig3highres.jpg" src="/files/ftp_upload/51154/51154fig3.jpg" /> 
Figure 3. Transduction of NPCs with lentiviruses expressing reporter genes (e.g. GFP. A and B, Phase contrast images of neurospheres (A) and differentiating NPCs (B) that have been transduced with a 3rd generation lenti-GFP vector. Flow cytometry analysis of the NPCs in A. C, Wild type (not transduced) and lenti-GFP transduced NPCs cultured as neurospheres are passaged every 4-5 days. Numbers of live and dead cells are collected and cumulative cell numbers plotted to generate a growth curve. The scale bar in A and B is 200 &#956;m. Data in C are mean cumulative number of cells &#177; SD (n=3).
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig4highres.jpg" src="/files/ftp_upload/51154/51154fig4.jpg" /> 
Figure 4. Functional characterization of chronic EAE. A, Daily weight monitoring in MOG- and CFA-immunized C57Bl/6 mice over a total follow up of 40 dpi. B, Daily EAE score monitoring in MOG- and CFA-immunized C57Bl/6 mice over a total follow up of 40 dpi. Data are expressed as mean numbers &#177; SD (n=10 mice/group).
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig5highres.jpg" src="/files/ftp_upload/51154/51154fig5.jpg" /> 
Figure 5. Systemically injected NPCs migrate in the parenchyma of EAE mice. A-E, X-gal staining of vibratome-cut (70 &#181;m) brain (A) and spinal cord (B) tissue sections from EAE mice injected i.v. with syngenic NPCs, showing transplanted &#946;-gal+ cells (blue cells) persisting within perivascular CNS areas up to the end of the clinical follow up (106 dpi). C, &#946;-gal+ i.v.-injected NPCs (blue) persisting within perivascular CNS areas, retain a Nestin+ (brown) phenotype. D, Few migrating NPCs express NeuN+ (brown) as long as they move out of the perivascular area. E, X-gal staining in a representative spinal cord section from a sham-treated EAE mouse. Scale bars, 20 &#956;m. F-G, Analysis of tissues other than the CNS shows that i.c.- or i.v.-injected NPCs (blue cells) reach virtually all the organs (e.g. lung, liver, spleen, heart, gut, kidney) within 10 dpt (F), but are cleared from the same organs by 30 dpt (G). Figures 5A-E is reproduced from reference 5, while Figures 5F-G is reproduced from Pluchino et al.2
<img alt="Figure 6" fo:content-width="5in" fo:src="/files/ftp_upload/51154/51154fig6highres.jpg" src="/files/ftp_upload/51154/51154fig6.jpg" /> 
Figure 6. Schematic representation of the protocol for systemic injection of NPCs in mice with EAE. Main critical steps of the systemic injection of NPCs in mice with EAE: from the preparation of injectable CAM expressing single cell dissociated NPCs the cell culture room (A), to the injection into the tail vein of EAE mice at the peak of the effector/invasion disease phase (B), towards the in vivo detection of the injected NPCs that have targeted the CNS (C), to the in vivo tissue pathological studies allowing the study of the mechanisms of therapeutic plasticity of injected NPCs (D). In D, green cells in are oligodendrocytes, dark blue cells are axons, light blue cells are transplanted NPCs, orange cells are astrocytes, and red cells are endothelial cells.
<table>
	<tbody>
		<tr>
			<td>Score</td>
			<td>Locomotor Responses</td>
		</tr>
		<tr>
			<td>0</td>
			<td>Normal mouse. No overt signs of disease</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Limp tail: complete flaccidity of the tail, and absence of curling at the tip of the tail when a mouse is picked up</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>Hind limb weakness: the mouse shows occasional and brief trippings when walking on a waddling gait</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Limp tail and hind limb weakness: when placed on its back, the mouse cannot turn to its normal position</td>
		</tr>
		<tr>
			<td>2.5</td>
			<td>Partial hind limb paralysis: the mouse can no longer use hind limbs to maintain rump posture or walk but can still move one or both hindlimbs to some extent, forelimbs remain unaffected</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Complete hind limb paralysis: total loss of movement in hindlimbs. The mouse drags itself only on its forelimbs, which remain unaffected. Mice at this stage are given food on the cage floor, long sipper tubes, and daily subcutaneous injection of hydrating solutions to prevent dehydration. If mice develop sores or skin lesions that do not recover with treatment, they will be killed humanely</td>
		</tr>
		<tr>
			<td>3.5</td>
			<td>Forelimb weakness: when place on a vertical grid, the mouse is not able to climb and slowly falls</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Moribund state</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Mouse found dead or culled according to humane endpoints</td>
		</tr>
	</tbody>
</table>
Table 1. Five stage scale of clinical signs and ascending paralysis in EAE mice.

Watch this Scientific Journal Video about Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE at JoVE.com

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

systemic-injection-neural-stemprogenitor-cells-mice-with-chronic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

17.6K Views.  University of Cambridge, UK. The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Video: Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...