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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2005 as doi:10.1096/fj.05-4170fje. |
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,
,1
* Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation, Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena, Milan, Italy;
Centre of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy; and
IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy
1 Correspondence: Department of Neurological Sciences, University of Milan, IRCCS Foundation, Ospedale Maggiore, Policlinico Mangiagalli and Regina Elena, Padiglione Ponti, Via Francesco Sforza 35, Milan 20122, Italy. E-mail: giacomo.comi{at}unimi.it
SPECIFIC AIMS
Neural stem cells may offer a possible source of cells for transplantation in neurological diseases. The aim of this study was the isolation and characterization of a primitive neural stem cell population double positive for LewisX(LeX) and CXCR4(CX) antigens that possesses CNS-homing potential and extensive neuronal-repopulating capacity in vivo.
PRINCIPAL FINDINGS
1. Identification and isolation of CNS derived LeX(ssea-1)+/CXCR4+ (LeCX) cells
To identify and isolate putative neural stem cells (NSC), we performed immunohistochemistry and FACS analyses to examine single cell suspension from embryonic and adult neurospheres for the expression of the cell surface marker LeX and for the chemokine receptor CXCR4. Double positive Le+CX+ cells were 14.8 ± 3.4% in embryonic cultures and 12.6 ± 4.2% in adult cultures. FACS-selected positive fractions ranged from 70 to 98% purity (Fig. 1
). We demonstrated the coexpression of LeX and CXCR4 in the radial glia cells of the murine developing brain as well as sparse cells of the adult SVZ region (Fig. 1)
.
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2. Le+CX+ cells are self-renewing and can acquire multiple neural and non-neural phenotypes
To confirm that selected cells are NSC, we first tested whether individual cells are able to generate multipotent neurospheres. The frequency of stem cells is 1:4 Le+CX+ cells and 1:420 in Le–CX– cells in embryonic spheres, and 1:8 Le+CX+ and 1:580 in Le–CX– in adult spheres (P<0.001). Le+CX+ cells were present both in primary and secondary spheres. The multipotency of the expanded neurospheres derived from Le+CX+ cells was confirmed after in vitro differentiation, resulting in cells expressing markers for neurons, astroglial cells, or oligodendrocytes (Fig. 1)
.
Le+CX+ cells exposed to "neuronal priming" conditions generated cholinergic neurons as demonstrated by the expression of Islet-1 and choline acetyl transferase (ChAT).
In the presence of appropriate inductive signals, Le+CX+ cells also generated myogenic and endothelial cells.
Le+CX+ populations, when cocultured with murine myoblasts or in the presence of Wnt3a (without coculture) acquired a striated skeletal muscle phenotype, expressing specific myogenic markers as confirmed by immunocytochemistry, Western blot, and real-time RT-PCR analyses.
In vivo, after transplantation into skeletal muscle, Le+CX+ cells contributed to muscle regeneration and to the restoration of dystrophin expression in mdx mice, an animal model of Duchenne muscular dystrophy.
To assure that Le+CX+ do differentiate into endothelial cells without cell fusion, we cultured them with endothelial cells separated by a trans-well membrane. FISH analysis for sex chromosomes excluded cell fusion events.
We demonstrated that Le+CX+, in culture conditions for ES and in the presence of LIF, expressed Oct4 and Rex1 transcripts. Le+CX+ cells derived from mice carrying the green fluorescent protein (GFP) under the control of the Oct4 promoter generated few Oct4-GFP-positive cells.
3. CXCR4-SDF pathway enhances LeCX chemotaxis, endothelial transmigration, proliferation, and survival
To directly estimate the chemoattractant effects of SDF on Le+CX+ fractions, we performed a Boyden chamber-based migration assay and endothelial transmigration analysis.
SDF stimulation resulted in a significant chemoattractant and transmigration effect on Le+CX+ fractions in a dose-dependent manner (P<0.001), while migration was significantly reduced by the specific CXCR4 inhibitor AMD3100. SDF significantly increased proliferation of the Le+CX+ (P<0.005) and protected this cell fraction from induced apoptosis (P<0.005).
SDF effects of Le+CX+ are mediated by the increased phosphorylation of p38 MAP kinase and ERK1/2, protein kinases as shown by Western blot analysis.
4. Intracerebroventricular transplantation of LeCX cells
To determine whether Le+CX+ could generate neurons in vivo, we transplanted these cells into the cerebral ventricles of newborn mice and adult mice, using both embryonic and adult LeCX cells. To trace the grafted cells, two donor mice strains were used: ß-actin-GFP and Thy1-YFP transgenic mice, which express the gene reporter ubiquitously and only in a subset of neurons, respectively. Two months after transplantation, many GFP/YFP cells were found in the gray and white matter, including the upper cerebral cortex. In Le+CX+ transplanted animals, quantification of incorporated GFP cells in the three selected regions revealed significantly higher densities vs. Le–CX–, Le+CX– fractions, or unselected cells (P<0.001).
GFP/YFP-positive cells from LeCX-positive fractions exhibited various neuronal morphologies and positivity for neuron-specific markers NeuN, TuJ1, NF, MAP2 and neurotransmitters (glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), or ChAT).
In the CA1 pyramidal cell layer of the hippocampus, we detected GFP/YFP-positive cells with pyramidal morphologies and appropriate dendritic orientation. In the cortex, we also observed cells with the morphological features of pyramidal neurons displaying large angular somas and rich trees of branching and spine-bearing apical and basal dendrites. These cells showed vertical alignment, perpendicular to the cortical layers, with the apical dendrite extending toward the pial surface (Fig. 2
).
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Since these phenotypes had not been described after neural stem cell transplantation, we wondered whether cell fusion occurred between LeCX-positive cells and endogenous neurons. In female mice transplanted with male GFP/YFP transgenic LeCX, we detected no trace of Y chromosome in any GFP/YFP neuronal cells examined using FISH (n=1775 GFP and n=565 YFP cells analyzed in 12 recipients, 6 neonatal and 6 adult recipients). This study suggests that stable fusion did not occur.
5. Systemic transplantation of LeCX cells
After injection through the tail vein of adult C57BL/6 mice and coadministration into the brain of SDF, Le+CX+ cells migrated into cortical and subcortical regions. Many donor cells were found in the meningeal layer and perivascular areas. The majority of these cells are round-shaped, negative for hematopoietic markers, and positive for vimentin and nestin. Some cells presented a more complex ramified phenotype with neuritic extensions positive for neuronal antigens. FISH analysis for Y chromosome in sex-mismatched transplantation ruled out the cell fusion event as mechanism of neurons generation. Mice administered with Le–CX– and Le+CX– fractions (both from embryonic and adult cells) did not show any apparent presence of donor cells in the brain when intravenously transplanted, even after cytokine intrabrain injections. When Le+CX+ were exposed to SDF inhibitor AMD3100 before i.v. injection, no donor cells were observed in the recipient CNS.
CONCLUSIONS AND SIGNIFICANCE
Achieving efficient distribution of NSC throughout the CNS and robust generation of specific neurons is a major challenge for the development of cell-mediated therapy for neurodegenerative diseases.
This study demonstrates for the first time that double positive LeX and CXCR4 populations, derived from both embryonic and adult neurospheres, are highly enriched for NSC that possess CNS-homing potential and multilineage repopulating capacity in vivo. Extensive generation of region-specific neurons is achievable by means of intraventricular and, to a lower extent, intravenous administration of LeCX cells. Moreover, the potential of neurosphere cells to cross the endothelial barrier in vitro and in vivo resides within the Le+CX+ fraction. The double positive LeX/CXCR4 fraction contains the majority of clonogenic cells, while the negative fraction rarely generates neurospheres. It is important to note that Le+CX+ cells are not only able to self-renew but they can also differentiate into neurons and macroglia cells as well as in mesodermal and endodermal phenotypes.
The major transplantation efficiency in vivo is likely due to the high proliferation, survival, and migratory capacity of Le+CX+ that we also demonstrated in vitro. We showed that Le+CX+ could generate cells with the morphological characteristics of intrinsic pyramidal neurons, including elaborated apical and basal neuritic extensions and vertical alignment within appropriate cortical and hippocampal layers. These complex phenotypes have not been described previously. FISH analysis in sex-mismatched transplantation experiments supports that stable fusion between the donor cells and recipient neurons does not occur. Thus, the mechanism responsible for the neuron formation from the Le+CX+ is the direct differentiation pathway both after direct or systemic transplantation.
Current efforts are primarily focused on making gene therapy and stem cell-mediated repairs efficient enough for clinical therapy. The CNS-homing tendency of Le+CX+ and their ability, particularly when stimulated in vitro using appropriate signals, to differentiate into diverse mature neuronal phenotypes and form appropriate long-distance connections, suggest that isolation of specific stem cell populations could offer major advantages for neuronal replacement and brain repair.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4170fje;
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32% of cells positive for LeX (A) and 60% for CXCR4 (green histogram) (B). After FACS separation for both LeX and CXCR4, positive cells demonstrated a high grade of purity (red histogram). Blue histogram represents control. Cryostat sections of embryonic derived neurospheres stained for LeX (C) and CXCR4 (D) and merge (E). Immunohistochemical analysis of embryonic brain (E 13.5). Radial glial cells identified by the expression of RC2 antigen (G) also coexpress LeX (F). Colabeling of CXCR4 (J) and LeX (I) was detected in radial glia. In vitro, Le+CX+ generate cells that morphologically resemble radial glial cells, with long processes positive for RC2. L) GFP; M) RC2. Neurospheres generated from LeCX-positive cells give rise to both neuronal and glial lineages. O) E.LeCX-derived neurospheres from YFP transgenic mice that express YFP protein (green) only in neurons produce neuronal cells expressing TuJ1 (red) and more mature neurons double positive for TuJ1 and YFP (yellow cells). Macroglial differentiation was confirmed by the detection of astrocytic (GFAP) (P) and oligodendrocyte markers (O4) (Q). Nuclei are counterstained with DAPI. Under differentiative conditions E.Le+CX+ fractions acquire a complex morphology with long cytoplasmatic neuritic extensions. These cells are double labeled (T, W, Z) with YFP (R, U, X) and TuJ1 (S), NF (V), or MAP2 (Y). Scale bars: C–E, I–K, P) 200 µm; F–H) 150 µm; O) 100 µm; Q) 50 µm; R–T) 100 µm; U–Z) 40 µm.