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Keywords:

  • Neural stem cells;
  • Aldehyde dehydrogenase;
  • Self-renewal;
  • Differentiation properties

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Stem cells are undifferentiated cells defined by their ability to self-renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues, and an emerging idea is that they share common characteristics such as the presence of ATP-binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity, raising the hypothesis of a set of universal stem cell markers. In the present study, we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue, based on the expression of high levels of ALDH activity. Single-cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSClo) and bright ALDH (ALDHbr) activity was isolated. SSCloALDHbr cells are capable of self-renewal and are able to generate new neurospheres and neuroepithelial stem-like cells. Furthermore, these cells are multipotent, differentiating both in neurons and macroglia, as determined by immunocytochemistry and real-time reverse transcription–polymerase chain reaction analysis. To evaluate the engraftment potential of SSCloALDHbr cells in vivo, we transplanted them into mouse brain. Donor-derived neurons with mature morphology were detected in the cortex and subcortical areas, demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs, and improvement of the stem cell isolation protocol may be useful in the development of a cell-mediated therapeutic strategy for neurodegenerative diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Stem cells are multipotent cells able to self-renew and generate immature and differentiated cell populations by asymmetric division [1]. Stem cells or their derivatives have been proposed as a potential source of transplantable cells for cellular-mediated therapies for neurodegenerative diseases [2].

Stem cells have been isolated from almost all tissues, and recent evidence suggests that they share some common properties such as the presence of ATP-binding cassette (ABC) transporter G2 (ABCG2) [3, 4], along with high telomerase [5] and aldehyde dehydrogenase (ALDH) activity [5, 6]. It is thus legitimate to raise the question as to the existence of a set of universal stem cell markers. A possible hypothesis is the existence of a “stem cell state” partially determined by the stem cell niche, the environment in which the stem cell resides [7].

Neural stem cells (NSCs) are usually identified retrospectively by their ability to generate in vitro free-floating aggregates termed neurospheres. However, neurospheres are not a pure population of stem cells and contain only a low number of neural progenitors [812].

The heterogeneity of these cells suggests that they may not be equivalent in their “repopulating” and homing potential when used as a source for cell transplantation. Recent studies have attempted to isolate NSCs from the adult mouse forebrain, using different strategies based on cell size combined with a number of negative selection criteria [13], the exclusion of the DNA binding dye Hoechst 33342 (side population) [14], or the expression of the surface antigen Lewis X [15] and CD133 in human NSCs [16]. However, stem cell isolation procedures based on complex fluorescence-activated cell sorting (FACS) analysis may not be easily reproduced, which is a limitation for wider experimental or prospective clinical applications. The identification of a cell surface marker is a valuable approach, but the surface phenotype of stem cells may remain stable despite a decline in functional activity as observed in the hematopoietic field [17]. For these reasons, we decided to investigate a simpler and more reliable isolation method for primitive NSCs. To identify primitive NSCs, we adopted an approach based on cell exposure to fluorescent ALDH substrate BODIPY aminoacetaldehyde (BAAA) – Aldefluor – consisting of an aminoacetaldehyde moiety bonded to the BODIPY fluorochrome, followed by flow cytometric analysis because ALDH is expressed at high levels in stem cells [5, 18, 19] as compared with more differentiated cells.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

NSC Isolation and Culture

NSCs were isolated from the forebrain germinal zones of 13.5 murine embryos and from adult forebrain subventricular dissection as previously described [20]. The cells were grown in neurobasal medium (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) containing N2 (Invitrogen), epidermal growth factor (EGF) (20 ng/ml; Sigma, St. Louis, http://www.sigmaaldrich.com), basic fibroblast growth factor (bFGF) (20 ng/ml; Sigma), penicillin (100 U; Invitrogen), and streptomycin (100 mg/ml; Invitrogen). The cultures were passaged every 5–7 days. Cells used for separation were passaged three to five times.

For donor cells for in vitro and in vivo experiments, we used either C57BL/6 mice or transgenic mice expressing a specific gene reporter. B6.Cg-TgN(Thy1-YFP)16Jrs mice express high levels of a spectral variant of green fluorescence protein (GFP), yellow fluorescence protein (YFP), in motor and sensory neurons as well as in subsets of central neurons [21]. Genotyping was performed by polymerase chain reaction (PCR) as already described [21]. The transgenic construct contains a YFP gene under the direction of regulatory elements derived from the mouse Thy1 gene. These elements are composed of a 6.5-kb fragment obtained from the 5′ portion of the Thy1 gene, extending from the promoter to the intron following exon 4. Exon 3 and its flanking introns are absent. The deleted sequences are required for expression in non-neural cells but not in neurons. The remaining sequence is required for neuronal expression [21].

All transgenic animals were purchased from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). All animal experiments were performed according to institutional guidelines in compliance with national (The Italian Law no. 116 published at the Gazzetta Ufficiale, suppl 40, February 18, 1992, and Law no. 8, Gazzetta Ufficiale, July 14, 1994) and international law and policies (EEC Council Directive 86/609, OJ L358, 1 December 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996).

Aldefluor Cell Analysis and Flow Cytometry

Cells obtained from freshly dissociated brain (as described above, as starting tissue for neurosphere culture) or from neurospheres were suspended in Aldefluor assay buffer containing ALDH substrate, BAAA (1 μmol/l) for 30–60′ × 1 × 106 cells. After staining, cells were maintained in ice during all subsequent procedures.

In each experiment, a sample of cells was stained under identical conditions with 50 mmol/l of specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) as negative control. Flow cytometric sorting was conducted using a FACS Vantage SE (Becton Dickinson Immunocytometry System, Mountain View, CA, http://www.bd.com). Aldefluor fluorescence was excited at 488 nm, and fluorescence emission was detected using a standard fluorescein isothiocyanate (FITC) 530/30-nm band-pass filter. Low side scatter (SSClo) and high ALDH (ALDHbr) were selected.

In addition, SSCloALDHbr cells were separated from the neurospheres of Thy-1 YFP mouse embryos. Although the YFP and ALDH wavelengths are similar, we were able to separate neurospheres from Thy-1 YFP mice because Thy-1 YFP expression is detected only in mature neurons, whereas undifferentiated neurospheres do not express YFP. Furthermore, we set FACS separation analysis using Aldefluor-unlabeled Thy1-YFP neurospheres as negative control. Aldefluor analysis and FACS were performed as described for wild-type cells.

In some trials, the suspension was incubated for 20 minutes at 4°C with biotin-conjugated peanut agglutinin (PNA, 1:200; Vecta; Vector Labs, Burlingame, CA, http://www.vectorlabs.com) [13], rinsed twice with serum-free medium by centrifugation at 4°C followed by Cy-3 streptavidin for 20 minutes at 4°C, then rinsed twice with medium by centrifugation at 4°C [13].

Cell size was assessed by forward light scatter FACS analysis as previously described [13].

SSCloALDHbr Versus Non-SSCloALDHbr Cell Culture

SSCloALDHbr and non-SSCloALDHbr cells were plated at equivalent densities (ranging from one to 10 cells per μl, depending on the number of isolated cells) in the presence of EGF and bFGF [20]. Neurospheres and clones were counted after 10–14 days in vitro. The difference of NSC enrichment was evaluated by analysis of variance (ANOVA) followed by Tukey post hoc test. The experiments were performed in triplicate and repeated three times. To induce differentiation, SSCloALDHbr-derived cells were plated onto poly-l-lysine–coated four-well plates (Nunc, Rochester, NY, http://www.nuncbrand.com) in neurobasal medium supplemented with B-27 supplement (In-vitrogen) without growth factors and with 1% fetal bovine serum (Sigma) for 7 days. B-27 is a serum-free supplement designed for the long-term viability of central nervous system (CNS) neurons.

Immunocytochemistry on Cell Culture

Cells were fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature. After rinsing with phosphate-buffered saline (PBS) and preincubation in a mixture of 5% normal serum and 0.25% Triton X-100 in PBS, cells were incubated with the primary antibodies (see below) overnight at 4°C. The following proteins were evaluated: nestin (mouse monoclonal, 1:200; Chemicon, Temecula, CA, http://www.chemicon.com), vimentin (mouse monoclonal, 1. 200 Novocastra), SOX2 (rabbit, 1:200, Chemicon), Musashi (rabbit, 1:200, Chemicon), NG2 (rabbit, 1:200, Chemicon), β-III-tubulin (TuJ-1, mouse monoclonal, 1:200; Chemicon), neurofilament (NF) M and H phosphorylated (mouse monoclonal, 1:200; Chemicon), nuclear specific neuronal antigen NeuN (mouse monoclonal, 1:100; Chemicon), mouse monoclonal anti–microtubule-associated protein 2 (MAP2; 1:100 dilution; Sigma-Aldrich), mouse Cy3-conjugated glial fibrillary acid protein (GFAP; 1:400 dilution; Sigma), and oligodendrocyte antigen (O4; mouse monoclonal, 1:100; Chemicon). Alexa-488 rabbit polyclonal antibody was used to recognize the GFP (1:400; Molecular Probes, Eugene, OR, http://probes.invitrogen.com). After repeated rinses in PBS, appropriate secondary antibodies (FITC and phycoerythrin R-PE or tetramethylrhodamine, 1:100; Dako, Carpinteria, CA, http://www.dako.com) were applied (1 hour in the dark at room temperature). Immunocytochemistry was also performed by omitting the incubation with primary antibodies as negative controls.

To evaluate differentiation properties, 50–100 primary neurospheres from SSCloALDHbr cells were differentiated and examined for the expression of neuronal (MAP2), astrocytic (GFAP), and oligodendrocyte (O4) markers as described above. The number of positive cells was estimated and expressed as a percentage of all examined cells. The trials were repeated three times. Data were analyzed by ANOVA and Tukey post hoc test.

Real-Time Reverse Transcription–PCR

For the evaluation of neuronal transcripts, total RNA was extracted from SSCloALDHbr cells cultured in neuronal growth and differentiation medium. 3T3 cells were used as negative controls. Total RNA was prepared using the EUROzol isolation kit (Euroclone, Pero, Italy, http://www.euroclone.net). Reverse transcription (RT) was carried out as follows: Two micrograms of total RNA from each sample was reverse-transcribed using random primers by the Ready-To-Go “You-Prime First-Strand Beads” kit (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) according to the manufacturer's recommendations. One microliter of the generated cDNA was used as template for each reaction in real-time quantitative PCR analysis, using Assays-on-Demand gene expression products in an ABI PRISM 7700 Sequence Detection System (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com). The following gene expression assays were performed: PAX6: Mm00443072_m1, nestin: Mm00450205_ m1, TuJ1: Mm00727586_s1, and GFAP: Mm00546086_m1. Eukaryotic 18S ribosomal RNA (Hs99999901_s1) was used as endogenous control to normalize all the above target genes; to determine relative quantification, the comparative threshold cycle method was used. The fold change in gene expression profile was compared to unseparated embryonic neurospheres.

All Assays-on-Demand gene expression products consist of target assays to amplify and detect expression of specific RNA sequences.

Cell Transplantation and Tissue Analysis

SSCloALDHbr cells from Thy1-YFP mice were used for transplantation after FACS selection.

Cells were transplanted into the lateral ventricles of anesthetized 1- to 2-day-old mouse pups and adults (6–8 weeks old) as previously described [22]. Two to three microliters of cell suspension (100,000 viable cells/μl) were slowly injected.

Two months after transplantation, the animals were sacrificed, perfused, and fixed with 4% PFA in PBS (pH 7.4).

The brain was isolated, immersed in PFA solution for 1 hour and then in sucrose 20% solution in PBS (pH 7.4) overnight, and frozen in Tissue-Tek OCT compound (Sakura, Torrance, CA, http://www.sakura-americas.com) with liquid nitrogen. The tissues were cryosectioned and mounted on gelatinized glass slides. Cerebral tissue was cut in the coronal plane from the frontal lobes (10 μm or 50 μm for stereological cell count). All CNS sections were blocked with 1% fetal calf serum in PBS and permeabilized with 0.25% Triton X-100.

Sections were processed for multiple markers to determine the cellular phenotype of YFP-labeled cells. Primary antibodies were added overnight at 4°C at dilutions of 1:200 for NeuN (mouse monoclonal antibody; Chemicon), 1:200 for NF-M and H phosphorylated (mouse monoclonal antibody; Chemicon), 1:200 for TuJ1 (mouse monoclonal antibody; Chemicon), 1:200 for MAP2 (mouse monoclonal antibody; Sigma-Aldrich), 1:200 for nestin (mouse monoclonal, Chemicon), 1:100 for rabbit anti–choline acetyl transferase (anti-ChAT) (Chemicon); 1:500 for rabbit anti–tyrosine hydroxylase (TH; Chemicon), 1:100 for anti-glutamic acid decarboxylase (anti-GAD 67) (mouse monoclonal; Chemicon), 1:300 for anti-synapsin I (mouse monoclonal; Chemicon), and 1:300 for anti-synaptotagmin (mouse monoclonal; Chemicon).

When unconjugated primary antibody was used, R-PE or Cy3 anti-mouse and anti-rabbit (1:100; DAKO) was used for 1 hour at room temperature as secondary antibody.

Anti-GFP antibody rabbit serum Alexa-488 (1:400 dilution; Molecular Probes) was used to reveal YFP positivity in double immunostaining.

Co-expression of YFP- and tissue-specific markers was evaluated by conventional fluorescence microscope (Zeiss Axiophot, Jena, Germany, http://www.zeiss.com) and by laser confocal scanning (Leica TCS SP2 AOBS, Heerbrugg, Switzerland, http://www.leica.com) microscopic analysis.

The criteria for scoring were a clear double staining, with homogeneous YFP expression and intact cellular morphology.

To obtain an unbiased stereological estimation of double-positive cells, optical dissectors and random sampling were used. For quantification of donor neurons, a systematic random series of every fifth section throughout the entire neocortex, striatum, and hippocampus (anatomical region boundaries were defined according to Franklin and Paxinos [23]) was selected, collecting 10 to 12 sections per animal for the hippocampus and striatum and 25–30 sections per animal for the neocortex. Every first section (50 μm) from one in five series was sampled using a random starting point.

The numerical density of neurons was then estimated by counting the number of neurons within three-dimensional optical dissectors that were systematically spaced at random throughout the selected brain areas. Optical dissectors sized 100 ×70 ×15 μm were randomly sampled, and the number of positive cells in each dissector was quantified. The mean number of dissectors sampled per section was 8.2 ± 1.1. The density was calculated by dividing the total number of YFP cells by the total volume of optical dissectors. Data are expressed as the number of cells per cubic millimeter and presented as means ± SD. Data were statistically analyzed by ANOVA and Fisher post hoc test. A significance level of 5% was assumed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Isolation of SSCloALDHbr NSCs

High ALDH activity is present in several stem cell types. In fact, staining with a fluorescent substrate for ALDH has been previously described to identify primitive hematopoietic stem cells [18].

To determine whether this strategy may be useful for the isolation of NSCs too, embryonic day 13.5 (E13.5) and adult dissociated brain and neurospheres were exposed to Aldefluor and then analyzed by flow cytometry for SSC properties and intracellular fluorescence staining intensity.

In each experiment, a sample of cells was stained simultaneously with Aldefluor and DEAB—an inhibitor of ALDH—to specifically identify the ALDHbr population (Fig. 1A).

A population of cells with SSClo property and high levels of ALDH staining, normally absent with DEAB, was detected (Fig. 1B). Brightly fluorescent cells amounted to 0.2%–0.4% of freshly dissociated tissues. An analysis of dissociated neurospheres revealed an increase in the SSCloALDHbr fraction, accounting for 4.21% ± 1.56% of embryonic and 2.82% ± 1.27% of adult neurospheres. Bright cells were sorted by FACS and positive fractions ranged from 80%–99% of purity.

SSCloALDHbr and negative cells were collected separately and plated in the presence of EGF and bFGF.

When observed by microscopy (Fig. 1D), SSCloALDHbr cells demonstrated a relatively homogenous round-shaped morphology. To characterize their phenotype, we double-stained the isolated cells with neuroectodermal markers.

In fact, at immunocytochemistry, the majority of cells expressed high levels of nestin, SOX2 (Fig. 2A), and Musashi antigens (p < .001 compared with differentiated cells), demonstrating their stem cell characteristics, whereas only a minimal fraction of these cells were positive for TuJ1, GFAP, and NG2 (GFAP and TuJ1, p < .001; NG2, p < .005, compared with differentiated cells) (Fig. 3A, 3C, 3E, 3G).

Real-time RT-PCR analysis confirmed the expression of high levels of nestin and the low expression of GFAP and TuJ1 in statistically significant terms, p < .001. Furthermore, we demonstrated that the expression level of the PAX6 transcript did not vary constantly throughout the differentiation process (Fig. 3B, 3D, 3F, 3H).

Examination of SSCloALDHbr cells revealed a uniform population of well circumscribed small cells averaging 7 μm in size. Indeed, sorted SSCloALDHbr cells were analyzed for PNA binding, resulting in a percentage of 32.4% ± 6.6% of PNA high cells (Fig. 1C).

SSCloALDHbr Cells Display Self-Renewal and Multipotent Properties

We evaluated the self-renewal capacity and multipotency of SSCloALDHbr cells in vitro by growing them at clonal density. One of seven single SSCloALDHbr embryonic cells and one of 11 single SSCloALDHbr adult cells generated spheres, showing a significant enrichment compared with the negative population and unselected neurospheres (p < .0001). Furthermore, one of 10 single SSCloALDHbr embryonic cells and one of 50 single adult cells generated flat clones that contained a hundred to a thousand cells. One of 16 cells deriving from dissociated embryonic tissue and one of 25 cells from adult tissue generated spheres (vs. the negative population and unselected neurospheres: p < .0001) (Table 1). Two of 100 embryonic cells versus only one of 1,000 adult tissue cells generated flat clones.

Clones and spheres can be passaged and can generate secondary and tertiary clones/spheres (80% of embryonic and 70% of adult cells).

Under basal conditions, all neurospheres deriving from SS-CloALDHbr cells expressed Sox2 (92.7% ± 4.6%) (Fig. 2B), Musashi (88.9% ± 4.8%) (Fig. 2C), nestin (97.6% ± 2.2%) (Fig. 2D), and vimentin (94.6% ± 4.3%) (this percentage refers to primary neurospheres from SSCloALDHbr deriving from embryonic cells) (p < .001 vs. differentiated cells). GFAP immunoreactivity was present in seven out of 115 neurospheres; within these positive spheres, GFAP cells represented an 11.6% ± 4.3% whereas, considering all the analyzed cells, the mean GFAP proportion was 0.71% ± 2.96%. The NG2 expression was detected in three out of 70 spheres (NG2-positive cells: 6.6% ± 2.2%; 0.28% ± 1.39% of all cells). GFAP staining and NG2 staining were mutually exclusive. TuJ1-positive cells were less frequently detected compared with the GFAP and NG2 positivity (four out of 98 spheres, 6.5% ± 2.3%; 0.26% ± 1.35% of all cells). A schematic diagram showing the immunoprofile of the primary spheres for each cell/tissue source is provided in Figure 3A, 3C, 3E, and 3G. The immunoprofile of the undifferentiated secondary and tertiary neurospheres was similar to that of primary spheres.

Real-time RT-PCR analysis showed the expression of high levels of nestin and PAX6 and, to a lower extent, TuJ1 and GFAP (Fig. 3B, 3D, 3F, 3H).

Individual clones and spheres were allowed to differentiate after plating and were cultured for 5–7 days in differentiative conditions, then processed for indirect immunocytochemistry with antibodies against neuronal (Fig. 2G–2I), astrocytic (Fig. 2E), and oligodendrocytic markers (Fig. 2F). This analysis demonstrated that the majority of SSCloALDHbr-derived neurospheres from both embryonic and adult sources generated colonies that contain neurons, astrocytes, and oligodendrocytes, indicating that the sorted clone-initiating cells are multipotent. Thus, the original SS-CloALDHbr-initiating cells own both self-renewal capacity and multipotency, which is suggestive of their NSC nature.

During differentiation, the SSCloALDHbr-derived neurospheres downregulated the stem markers nestin, Sox2, and Musashi in a statistically significant way (p < .001) compared with freshly separated cells (T0) and spheres, whereas they upregulated the neuronal differentiative (TuJ1), astroglial (GFAP), and oligodendroglial (NG2) antigens (GFAP and TuJ1: p < .001; NG2: p < .005) (Fig. 3A, 3C, 3E, 3G).

Real-time RT-PCR analysis confirmed the increased expression of differentiated neuroectodermal transcripts such as TuJ1 and GFAP (Fig. 3B, 3D, 3F, 3H) (p < .001, differentiated cells vs. freshly separated cells [T0] and spheres).

In Vivo Cell Transplantation of SSCloALDHbr Cells into the Lateral Brain Ventricle of Neonatal Mice

To assess the ability of SSCloALDHbr cells to engraft and differentiate into neurons in different regions of the host CNS, SSCloALDHbr cells isolated from embryonic neurospheres of Thy1-YFP mice were injected into the lateral ventricles of neonatal mice. In all recipient animals, transplanted cells were detected in the host brains (Figs. 4, 5).

In particular, Thy1-YFP transplanted cells had incorporated into the gray and white matter of the brain, migrating from the cerebral ventricles within 2 months after delivery. They migrated into the midbrain area surrounding the cerebral aqueduct and were efficiently engrafted into the hippocampus, thalamus, and hypothalamus areas. Thy1-YFP neurons were also detected in the striatum, corpus callosum, and white matter as well as in olfactory bulbs. Less frequently, donor-derived neurons incorporated into the cerebellum, pons, and medulla oblongata. Interestingly, YFP cells had also reached the upper and lower cerebral cortical layers, confirming their extensive migratory potential.

The activation of Thy1-neuron–specific transgene observed in several brain regions indicated that the donor cells differentiated into neurons in the recipient. Many YFP-positive cells, traced by green fluorescence, showed cellular profiles resembling highly mature, morphologically differentiated cells with complex and long neuritic extensions. Some cells displayed an immature phenotype with short cytoplasmic branches or a bipolar shape.

In most instances, grafted cells incorporated into the host parenchyma as single-scattered elements, particularly in cortical areas. They were frequently gathered in cell cluster groups, often near the ventricles, and also in the host parenchyma and in the surface of brain hosts. The detected YFP signal within long cytoplasmic extensions represents colonies of differentiated donor-derived neurons. Furthermore, YFP-positive neurons in the neocortex, thalamus, and striatum exhibited various neuronal morphologies, including multipolar neurons, whereas donor-derived neurons resembling interneurons were found in the hippocampal areas, including CA1, CA3, subiculum, and sub-granular zones of the dentate gyrus, thus suggesting a precise site-specific differentiation (Fig. 4).

The overall YFP-neuron densities were 198.6 ± 44.4/mm3 (cortex), 307.6 ± 52.6/mm3 (striatum), and 603.5 ± 87.7/mm3 (hippocampus).

Moreover, the donor-derived neurons remarkably generated numerous axons projecting within long distances into the host brain (Fig. 4A, 4B). Fibers were detected in both the gray and white matter – rostrally in the cortex and olfactory bulbs and caudally in the cerebral peduncle. YFP-positive neurons were tested for the expression of cell type-specific markers in order to evaluate the differentiation potential of transplanted cells. Donor-derived YFP neurons expressed neuron-specific markers like NeuN, NF, TuJ1, and MAP2, thus confirming that ALDH cells acquire neuronal phenotype 2 months after transplantation.

Confocal three-dimensional reconstruction images clearly showed co-localization of YFP and neuronal markers in the same cells (Fig. 5C–5I).

Double immunohistochemistry shows the presence of different neurotransmitter subtypes in donor cells. Overall, 32.6% ± 8.8% of the YFP-positive neurons expressed GAD-67, the rate-limiting enzyme for GABA synthesis, indicating a GABAergic phenotype (Fig. 5M–5O).

A smaller cell population was immunoreactive for TH (6.7% ± 4.5%) or ChAT (15.0% ± 5.3%) (markers for catecholaminergic and cholinergic neurons, respectively) (Fig. 5J–5L, 5P–5R).

GABA-immunoreactive neurons were localized in the hippocampus, striatum, and cortex.

The potential establishment of synapses was confirmed by the number of spines observed on the dendrites of donor-derived neurons (Fig. 4C). The functional study of derived neurons was completed by the immunohistochemical analysis of synaptic vesicle proteins such as synapsin I (Fig. 4D–4F) and synaptotagmin, which were detected near and inside the newly formed YFP neurons by confocal analysis.

Transplants of SSCloALDHbr Cells into the Lateral Brain Ventricle of Adult Mouse Recipients

SSCloALDHbr cells implanted into the cerebral ventricles of adult mouse recipients migrated as a single cell into a variety of brain regions, with a distribution pattern similar to that observed in neonatal mice. Indeed, the donor cell density decreased with increasing distance from the ventricular wall. Donor-derived neurons incorporated into the subcortical regions of the striatum, hippocampus, olfactory bulb, septum, thalamus, hypothalamus, into the midbrain regions and the corpus callosum, white matter, and cortical layers (Fig. 6A–6E).

Stereological analysis demonstrated that the density of donor-derived YFP cells was 132.6 ± 38.8/mm3 (cortex), 186.8 ± 64.6/mm3 (striatum), and 396.6 ± 56.6/mm3 (hippocampus), a density significantly lower than that observed in neonatal recipients (p < .001).

Integrated YFP-positive neurons presented phenotypes varying from bipolar immature migrating neuroblasts to complex multipolar neurons in the hippocampus, striatum, and neocortex.

Neuronal-specific markers, such as NeuN, TuJ1, MAP2, NF, along with the YFP protein, were used in immunohistochemistry to define the phenotypic characteristics of the detected engrafted cells (Fig. 6C–6H). Interestingly, some cells expressed neurotransmitters like GAD (28.6% ± 4.5%) (Fig. 6I–6K), TH (6.6% ± 3.2%), and ChAT (14.3% ± 4.6%). In particular, a fraction of donor-derived neurons, integrated throughout the deeper and upper cortical layers of the adult mouse neocortex, was also positive for anti-GAD immunostaining, indicating that they differentiated into GABAergic phenotype. Furthermore, GAD-positive Thy1-YFP interneurons were observed in the striatum and hippocampus. The frequency of the neuronal spines from the dendrites confirmed the integration of donor cells within the host's neuronal network. The neoformation of synaptic contacts was also demonstrated by the detection of synaptic vesicle proteins [24] such as synapsin I and synaptotagmin (Fig. 6B) in immunohistochemical reactions. In fact, these synaptotagmin-positive vesicles colocalized with the YFP-positive signal of engrafted cells. Therefore, we suggest that transplanted cells present a high level of functional integration developing pre- and postsynaptic contacts with the host's tissue neurons.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

The relative rarity of primordial NSCs and the absence of stem cell–specific markers restrict their identification and isolation, limiting basic biological studies and therapeutic applications. To overcome this hurdle, we used a strategy developed for the isolation of hematopoietic stem cells that involves FACS analysis on the basis of the high ALDH activity of stem cells.

In this study, we demonstrated that cells with low SSC and high levels of ALDH activity (SSCloALDHbr) could be identified and isolated from freshly dissociated murine brain tissue and neurospheres by Aldefluor staining and FACS analysis.

Flow cytometric analysis revealed that neurosphere cultures contain a much higher percentage of cells that stain brightly with Aldefluor, compared with freshly dissociated tissues. However, cells sorted from neurospheres give rise to a population with essentially the same characteristics as freshly isolated brain cells, which may probably be attributed to the fact that neurospheres are already enriched with NSCs [25]. On the other hand, cells derived from fresh tissue may be affected by the dissociation procedure.

Furthermore, ALDH expression is much more consistent in embryonic tissues than in adult tissues. Non–ALDHbr-derived cells did not present stem cell properties of self-renewal and multipotentiality. Therefore, these cells are likely to be progenitor cells, and not NSCs [26].

The SSCloALDHbr population is relatively homogeneous as suggested by the evaluation of cell size, both by FACS analysis and direct microscopic observation. Furthermore, these cells express high levels of stem cell markers such as nestin, SOX2, and Musashi, confirming their primitive phenotype.

Rietze et al. [13] reported the isolation of a pluripotent NSC population from the adult mouse brain. The selection criteria included a cell diameter larger than 12 μm and a low expression level of PNA-binding activity. A twofold enrichment in NSC isolation was obtained when cells were sorted on the basis of size alone (>12 μm), whereas sorting for PNAlo cells resulted in an additional 22-fold enrichment in this population. These data are partially in contrast with findings by Kim and Morshead [14], who isolated NSCs using the side population analysis. In their experiments, side population NSCs presented 36% of PNAhi positivity, whereas more than 95% of the neurospheres were detected in the PNAhi-expressing population. Indeed, the 50% increase in stem cell frequency within the large cell fraction can be entirely attributed to clumping during sorting.

In the present work, the examination of SSCloALDHbr cell dimension at the forward scatter revealed a uniform population of small cells. Indeed, sorted SSCloALDHbr cells expressed a subpopulation of PNAhi cells in a proportion similar to that previously described in the side population NSCs.

SSCloALDHbr cells had a variety of properties of primitive NSCs, including the ability to generate both neurospheres and adherent colonies and to differentiate into multiple lineages. The acquisition of a neuroectodermal phenotype was confirmed by immunohistochemical and real-time RT-PCR analysis. The newly generated neurospheres presented a nearly homogenous phenotype, expressing almost uniformly NSC markers such as SOX2, Musashi, and nestin.

After transplantation into the neonatal brain, SSCloALDHbr cells displayed high engraftment and neurogenic potential. In vivo, these cells were widely distributed through cortical and subcortical areas and acquired different kinds of neuronal morphology, reaching—in the majority of cases—a complex mature phenotype. In particular, donor-derived cells appeared fully integrated into the host architecture, showing typical structure, orientation, and distribution of their processes. Furthermore, their long ramified dendritic processes, studded with spines, indicated a functional maturation of these grafted cells, suggesting their high grade of integration by developing pre- and postsynaptic contacts with the host's tissue neurons.

As observed in the case of the isolation of hematopoietic stem cells for hematological disorders, isolated SSCloALDHbr NSCs might present advantages over unfractionated cells for the development of a preclinical cell enrichment protocol for neurodegenerative diseases.

Various reports on hemopoietic stem cells showed that an ALDH subpopulation in bone marrow is highly enriched for hemopoietic progenitor cells able to renew and form colonies under appropriate conditions [6, 18]. In the CNS too, cells emitting bright fluorescence seem to represent a specific subpopulation. Indeed, our data suggest that the ALDHbr subpopulation contains both neurosphere-initiating cells and neuroepithelial-like cells in EGF/FGF culture conditions.

For the isolation of primitive NSCs, BAAA staining offers several potential advantages over current methods. First, it is a simple procedure with a high level of reproducibility. In addition, BAAA should not enter the nucleus or bind DNA. Consequently, the isolation of primitive cells by BAAA staining may be safer and less toxic than methods based on staining with dyes that bind to nucleic acids or require UV excitation, like side population analysis. Furthermore, Aldefluor as a cell viability dye makes it more reliable than surface marker selection–based methods that do not discriminate between viable and nonviable cells [27]. In fact, only viable cells with functional enzymatic activity and an intact membrane can retain the Aldefluor reaction product, whereas apoptotic and necrotic cells with leaky membranes are not counted. Because stem cells in many tissues may express high levels of ALDH, the SSCloALDHbr cell identification may represent a generically useful method for the isolation of somatic stem cells for future clinical applications.

Table Table 1.. Summary of enrichment profile for neural stem cells after FACS selection
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Figure Figure 1.. Identification, isolation, and differentiation of SSCloALDHbr cells from murine brain. (A, B): Representative flow cytometric analysis of ALDH activity on brain-derived cells. NSCs are selected according to FSC and SSC properties. NSCs incubated with Aldefluor substrate and the specific inhibitor of ALDH, DEAB, are used to establish the baseline fluorescence of these cells and to define the ALDHbr region (A). Incubation of NSCs with Aldefluor substrate in the absence of an inhibitor induces a shift in FL1 fluorescence defining the ALDHbr population (B). (C): PNA binding profile of SSCloALDHbr cells, revealing the presence of a PNAhi subpopulation (green histogram). Purple histogram represents control. (D): Microscopic examination (merge of optic and fluorescence microscopy) of SSCloALDHbr cells, demonstrating a relatively uniform population of small round cells positive for ALDH (green signal). Scale bar = 100 μm. Abbreviations: ALDH, aldehyde dehydrogenase; ALDHbr, bright aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; FSC, forward scatter; NSC, neural stem cell; PNA, peanut agglutinin; PNAhi, high peanut agglutinin; SSC, side scatter; SSClo, low side scatter.

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Figure Figure 2.. In vitro characterization of SSCloALDHbr cells. (A): The majority of sorted SSCloALDHbr cells express the neuroectodermal stem antigen SOX2 (red nuclear signal). (B): SSCloALDHbr cells in the presence of growth medium form spheres positive for SOX2 (red nuclear signal). (C): SSCloALDHbr cells in the presence of growth medium form spheres positive for Musashi (green). (D): SSCloALDHbr cells in the presence of growth medium form spheres positive for nestin (red). In differentiative conditions, these cells give rise to all three lineages. (E): Macroglial differentiation is confirmed by the detection of astrocytic markers (GFAP) (red). (F): Macroglial differentiation is confirmed by the detection of oligodendrocyte markers (O4) (red). (G–I): SSCloALDHbr-derived neurospheres from YFP transgenic mice that express YFP protein (green, [G]) only in neurons produce neuronal cells expressing TuJ1 (red, [H]). Among these cells, some are more mature neurons that are double-positive for TuJ1 and YFP (merged yellow cells, [I]). Nuclei are counterstained with DAPI (blue). Scale bar = 150 μm (A, B), 120 μm (C), 50 μm (D, E), 20 μm (F), 100 μm (G–I). Abbreviations: ALDH, aldehyde dehydrogenase; ALDHbr, bright aldehyde dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; DEAB, diethylaminobenzaldehyde; FSC, forward scatter; GFAP, glial fibrillary acid protein; NSC, neural stem cell; SSC, side scatter; SSClo, low side scatter; YFP, yellow fluorescence protein.

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Figure Figure 3.. Phenotypic characterization and quantification of SSCloALDHbr cells. Phenotypic evaluation by immunocytochemistry (A, C, E, G) and real-time RT-PCR (B, D, F, H) of SSCloALDHbr cells. (A): Cells separated from embryonic neurospheres. (B): Cells separated from embryonic neurospheres. (C): Cells separated from embryonic tissue. (D): Cells separated from embryonic tissue. (E): Cells separated from adult dissociated spheres. (F): Cells separated from adult dissociated spheres. (G): Cells separated from adult dissociated tissue. (H): Cells separated from adult dissociated tissue. (A, C, E, G): Results of immunocytochemical analysis of stem cell and neuroectodermal antigens in SSCloALDHbr cells. T0 cells and spheres highly expressed nestin, Sox2, and Musashi in a statistically significant way as compared with differentiated cells (p < .001). On the other hand, differentiated cells expressed neuronal differentiative antigens at more significant levels as compared with cells at T0 and sphere-derived cells (GFAP and TuJ1: p < .001; NG2: p < .005). (B, D, F, H): Real-time RT-PCR analysis confirmed the expression of high levels of nestin and the low expression of GFAP and TuJ1 at T0 and in SSCloALDHbr-derived spheres, whereas differentiated cells upregulated the neuronal differentiative (TuJ1) and astroglial (GFAP) antigens. The expression of the PAX6 transcript did not vary constantly throughout the differentiation process. The fold change in the gene expression profile was compared to unseparated embryonic neurospheres (square bars). Black bars represent early isolated SSC10ALDHbr cells (T0); black/white bars indicate SSCloALDHbr-derived spheres; white bars show differentiated cells. Square brackets combine drastically compared parameters. **p < .001; *p < .005. Abbreviations: ALDH, aldehyde dehydrogenase; ALDHbr, bright aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; FSC, forward scatter; GFAP, glial fibrillary acid protein; NSC, neural stem cell; RT-PCR, reverse transcription–polymerase chain reaction; SSC, side scatter; SSClo, low side scatter.

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Figure Figure 4.. Donor-derived axons projecting within long distances into the host brain and evidence of synapsis formation. (A): Widespread distribution of donor-derived neurons displaying long fibers in the hippocampal region. (B): Cortical area of neonatal recipients showing numerous donor-derived fibers extending in parallel and perpendicularly throughout the cortex. (C): Higher magnification of neuritic extensions demonstrating the presence of spines. (D): Double immunostaining of dendritic arborization for YFP. (E): Double immunostaining of dendritic arborization for synapsin. (F): Merged image. Nuclei are counterstained with DAPI blue signal. Scale bar = 180 μm (A), 150 μm (B), 30 μm (C), 90 μm (D–F). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; YFP, yellow fluorescence protein.

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Figure Figure 5.. Immunohistochemical analysis of SSCloALDHbr cells after in vivo transplantation into neonatal mouse brain. (A, B): At 2 months from transplantation, SSCloALDHbr-derived YFP neurons in the murine brain are distributed in cortical ([A] frontal cortex) and subcortical areas ([B] striatum). The expression of the specific neuronal gene reporter Thy1-YFP provides direct evidence that these cells are neurons. (C): In the cortical layer of the temporal cortex, double immunostaining (yellow signal) and three-dimensional confocal reconstruction of transplanted YFP cells (green) with TuJ1 neuronal marker (red) reveal the neuronal differentiation potentiality of SSCloALDHbr cells in vivo. (D–F): Confocal microscopy in the cortical layer shows a YFP donor-derived neuron ([D] green signal) expressing NeuN ([E] red signal, [F] merge). (G–I): A neuron double-positive for YFP ([G] YFP green signal) and NF ([H] red signal, [I] merge) is demonstrated by confocal microscopy in the cortex. A three-dimensional view is available online as a supplemental movie. (J–L): A donor-derived cell differentiates into neurotransmitter neuronal subtype as demonstrated by the double positivity of YFP ([J] green) and ChAT ([K] red, [L] merge). (M–R): At low magnification power, we showed YFP-positive cells ([M, P] green signals) fully integrated in the cortex that express GAD-67, the rate-limiting enzyme for GABA synthesis, indicating a GABAergic phenotype ([N] red signals), and TH ([Q] red signals). (O, R): merge, yellow signals, respectively. Nuclei are counterstained with DAPI blue signal. Scale bar = 70 μm (A), 80 μm (B, C), 40 μm (D–F), 80 μm (G–I), 70 μm (J–L), 150 μm (M–O), 120 μm (P–R). Abbreviations: ALDHbr, bright aldehyde dehydrogenase; ChAT, choline acetyl transferase; DAPI, 4′,6-diamidino-2-phenylindole; NF, neurofilament; SSClo, low side scatter; YFP, yellow fluorescence protein.

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Figure Figure 6.. Immunohistochemical analysis of SSCloALDHbr cells after in vivo transplantation into adult mouse brain. (A): Widespread distribution of SSCloALDHbr cells transplanted into the hippocampal area of adult mouse brain. (B): Merged confocal image of a YFP (green) donor-derived fully differentiated neuron displaying a branched neuritic extension double-positive for synaptotagmin (red) in the host's hippocampus (yellow: merge signal). (C–E): YFP donor-derived neurons ([C] green) with long projecting axons expressing NF neuronal marker ([D] red, [E] merge) integrated in the cortical areas. (F–H): A YFP neuron double-positive for YFP [F] green signal) and MAP2 ([G] red signal, [H] merge). (I–K): A fraction of YFP positive neurons ([I] green) was double-positive for GAD-67 ([J] red), suggesting a GABAergic phenotype ([K] merge). Nuclei are counterstained with DAPI blue signal. Scale bar = 180 μm (A), 90 μm (B, F–H), 100 μm (C–E, I–K). Abbreviations: ALDHbr, bright aldehyde dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole; NF, neurofilament; SSClo, low side scatter; YFP, yellow fluorescence protein.

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Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

This work was supported by research grants to G.P.C. from the Italian Ministry of Health, Ricerca Finalizzata 2004 “Studio di protocolli di terapia cellulo-mediata nelle patologie neurodegenerative e nelle distrofie muscolari,” MIUR (Ministero Istruzione Università e Ricerca Scientifica) Italian Ministery FIRB 2002 “Animali geneticamente modificati per lo studio di patologie neurodegenerative,” and Progetto a Concorso Ospedale Maggiore Policlinico 2003. We thank “Associazione Amici del Centro Dino Ferrari” for support.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information
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SC050217SuppFile1.mov1226KSupplemental Movie

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